@article{pmid37704610,

title = {De novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8},

author = {Anindya Roy and Lei Shi and Ashley Chang and Xianchi Dong and Andres Fernandez and John C Kraft and Jing Li and Viet Q Le and Rebecca Viazzo Winegar and Gerald Maxwell Cherf and Dean Slocum and P Daniel Poulson and Garrett E Casper and Mary L Vallecillo-Zúniga and Jonard Corpuz Valdoz and Marcos C Miranda and Hua Bai and Yakov Kipnis and Audrey Olshefsky and Tanu Priya and Lauren Carter and Rashmi Ravichandran and Cameron M Chow and Max R Johnson and Suna Cheng and McKaela Smith and Catherine Overed-Sayer and Donna K Finch and David Lowe and Asim K Bera and Gustavo Matute-Bello and Timothy P Birkland and Frank DiMaio and Ganesh Raghu and Jennifer R Cochran and Lance J Stewart and Melody G Campbell and Pam M Van Ry and Timothy Springer and David Baker},

doi = {10.1038/s41467-023-41272-z},

issn = {2041-1723},

year  = {2023},

date = {2023-09-01},

journal = {Nat Commun},

volume = {14},

number = {1},

pages = {5660},

abstract = {The RGD (Arg-Gly-Asp)-binding integrins αvβ6 and αvβ8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvβ6 and αvβ8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvβ6 and αvβ8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvβ6 and the αvβ8 integrins. In a lung fibrosis mouse model, the αvβ6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37433327,

title = {De novo design of protein structure and function with RFdiffusion},

author = {Joseph L Watson and David Juergens and Nathaniel R Bennett and Brian L Trippe and Jason Yim and Helen E Eisenach and Woody Ahern and Andrew J Borst and Robert J Ragotte and Lukas F Milles and Basile I M Wicky and Nikita Hanikel and Samuel J Pellock and Alexis Courbet and William Sheffler and Jue Wang and Preetham Venkatesh and Isaac Sappington and Susana Vázquez Torres and Anna Lauko and Valentin De Bortoli and Emile Mathieu and Sergey Ovchinnikov and Regina Barzilay and Tommi S Jaakkola and Frank DiMaio and Minkyung Baek and David Baker},

doi = {10.1038/s41586-023-06415-8},

issn = {1476-4687},

year  = {2023},

date = {2023-08-01},

journal = {Nature},

volume = {620},

number = {7976},

pages = {1089--1100},

abstract = {There has been considerable recent progress in designing new proteins using deep-learning methods. Despite this progress, a general deep-learning framework for protein design that enables solution of a wide range of design challenges, including de novo binder design and design of higher-order symmetric architectures, has yet to be described. Diffusion models have had considerable success in image and language generative modelling but limited success when applied to protein modelling, probably due to the complexity of protein backbone geometry and sequence-structure relationships. Here we show that by fine-tuning the RoseTTAFold structure prediction network on protein structure denoising tasks, we obtain a generative model of protein backbones that achieves outstanding performance on unconditional and topology-constrained protein monomer design, protein binder design, symmetric oligomer design, enzyme active site scaffolding and symmetric motif scaffolding for therapeutic and metal-binding protein design. We demonstrate the power and generality of the method, called RoseTTAFold diffusion (RFdiffusion), by experimentally characterizing the structures and functions of hundreds of designed symmetric assemblies, metal-binding proteins and protein binders. The accuracy of RFdiffusion is confirmed by the cryogenic electron microscopy structure of a designed binder in complex with influenza haemagglutinin that is nearly identical to the design model. In a manner analogous to networks that produce images from user-specified inputs, RFdiffusion enables the design of diverse functional proteins from simple molecular specifications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37384673,

title = {Hexasome-INO80 complex reveals structural basis of noncanonical nucleosome remodeling},

author = {Min Zhang and Anna Jungblut and Franziska Kunert and Luis Hauptmann and Thomas Hoffmann and Olga Kolesnikova and Felix Metzner and Manuela Moldt and Felix Weis and Frank DiMaio and Karl-Peter Hopfner and Sebastian Eustermann},

doi = {10.1126/science.adf6287},

issn = {1095-9203},

year  = {2023},

date = {2023-07-01},

journal = {Science},

volume = {381},

number = {6655},

pages = {313--319},

abstract = {Loss of H2A-H2B histone dimers is a hallmark of actively transcribed genes, but how the cellular machinery functions in the context of noncanonical nucleosomal particles remains largely elusive. In this work, we report the structural mechanism for adenosine 5'-triphosphate-dependent chromatin remodeling of hexasomes by the INO80 complex. We show how INO80 recognizes noncanonical DNA and histone features of hexasomes that emerge from the loss of H2A-H2B. A large structural rearrangement switches the catalytic core of INO80 into a distinct, spin-rotated mode of remodeling while its nuclear actin module remains tethered to long stretches of unwrapped linker DNA. Direct sensing of an exposed H3-H4 histone interface activates INO80, independently of the H2A-H2B acidic patch. Our findings reveal how the loss of H2A-H2B grants remodelers access to a different, yet unexplored layer of energy-driven chromatin regulation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37253357,

title = {Residue-level error detection in cryoelectron microscopy models},

author = {Gabriella Reggiano and Wolfgang Lugmayr and Daniel Farrell and Thomas C Marlovits and Frank DiMaio},

doi = {10.1016/j.str.2023.05.002},

issn = {1878-4186},

year  = {2023},

date = {2023-07-01},

journal = {Structure},

volume = {31},

number = {7},

pages = {860--869.e4},

abstract = {Building accurate protein models into moderate resolution (3-5 Å) cryoelectron microscopy (cryo-EM) maps is challenging and error prone. We have developed MEDIC (Model Error Detection in Cryo-EM), a robust statistical model that identifies local backbone errors in protein structures built into cryo-EM maps by combining local fit-to-density with deep-learning-derived structural information. MEDIC is validated on a set of 28 structures that were subsequently solved to higher resolutions, where we identify the differences between low- and high-resolution structures with 68% precision and 60% recall. We additionally use this model to fix over 100 errors in 12 deposited structures and to identify errors in 4 refined AlphaFold predictions with 80% precision and 60% recall. As modelers more frequently use deep learning predictions as a starting point for refinement and rebuilding, MEDIC's ability to handle errors in structures derived from hand-building and machine learning methods makes it a powerful tool for structural biologists.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37149653,

title = {Improving de novo protein binder design with deep learning},

author = {Nathaniel R Bennett and Brian Coventry and Inna Goreshnik and Buwei Huang and Aza Allen and Dionne Vafeados and Ying Po Peng and Justas Dauparas and Minkyung Baek and Lance Stewart and Frank DiMaio and Steven De Munck and Savvas N Savvides and David Baker},

doi = {10.1038/s41467-023-38328-5},

issn = {2041-1723},

year  = {2023},

date = {2023-05-01},

journal = {Nat Commun},

volume = {14},

number = {1},

pages = {2625},

abstract = {Recently it has become possible to de novo design high affinity protein binding proteins from target structural information alone. There is, however, considerable room for improvement as the overall design success rate is low. Here, we explore the augmentation of energy-based protein binder design using deep learning. We find that using AlphaFold2 or RoseTTAFold to assess the probability that a designed sequence adopts the designed monomer structure, and the probability that this structure binds the target as designed, increases design success rates nearly 10-fold. We find further that sequence design using ProteinMPNN rather than Rosetta considerably increases computational efficiency.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36859493,

title = {Automatic and accurate ligand structure determination guided by cryo-electron microscopy maps},

author = {Andrew Muenks and Samantha Zepeda and Guangfeng Zhou and David Veesler and Frank DiMaio},

doi = {10.1038/s41467-023-36732-5},

issn = {2041-1723},

year  = {2023},

date = {2023-03-01},

journal = {Nat Commun},

volume = {14},

number = {1},

pages = {1164},

abstract = {Advances in cryo-electron microscopy (cryoEM) and deep-learning guided protein structure prediction have expedited structural studies of protein complexes. However, methods for accurately determining ligand conformations are lacking. In this manuscript, we develop EMERALD, a tool for automatically determining ligand structures guided by medium-resolution cryoEM density. We show this method is robust at predicting ligands along with surrounding side chains in maps as low as 4.5 Å local resolution. Combining this with a measure of placement confidence and running on all protein/ligand structures in the EMDB, we show that 57% of ligands replicate the deposited model, 16% confidently find alternate conformations, 22% have ambiguous density where multiple conformations might be present, and 5% are incorrectly placed. For five cases where our approach finds an alternate conformation with high confidence, high-resolution crystal structures validate our placement. EMERALD and the resulting analysis should prove critical in using cryoEM to solve protein-ligand complexes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36586407,

title = {Structural basis underlying specific biochemical activities of non-muscle tropomyosin isoforms},

author = {Muniyandi Selvaraj and Shrikant B Kokate and Gabriella Reggiano and Konstantin Kogan and Tommi Kotila and Elena Kremneva and Frank DiMaio and Pekka Lappalainen and Juha T Huiskonen},

doi = {10.1016/j.celrep.2022.111900},

issn = {2211-1247},

year  = {2023},

date = {2023-01-01},

journal = {Cell Rep},

volume = {42},

number = {1},

pages = {111900},

abstract = {The actin cytoskeleton is critical for cell migration, morphogenesis, endocytosis, organelle dynamics, and cytokinesis. To support diverse cellular processes, actin filaments form a variety of structures with specific architectures and dynamic properties. Key proteins specifying actin filaments are tropomyosins. Non-muscle cells express several functionally non-redundant tropomyosin isoforms, which differentially control the interactions of other proteins, including myosins and ADF/cofilin, with actin filaments. However, the underlying molecular mechanisms have remained elusive. By determining the cryogenic electron microscopy structures of actin filaments decorated by two functionally distinct non-muscle tropomyosin isoforms, Tpm1.6 and Tpm3.2, we reveal that actin filament conformation remains unaffected upon binding. However, Tpm1.6 and Tpm3.2 follow different paths along the actin filament major groove, providing an explanation for their incapability to co-polymerize on actin filaments. We also elucidate the molecular basis underlying specific roles of Tpm1.6 and Tpm3.2 in myosin II activation and protecting actin filaments from ADF/cofilin-catalyzed severing.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36323866,

title = {StarMap: a user-friendly workflow for Rosetta-driven molecular structure refinement},

author = {Wolfgang Lugmayr and Vadim Kotov and Nikolaus Goessweiner-Mohr and Jiri Wald and Frank DiMaio and Thomas C Marlovits},

doi = {10.1038/s41596-022-00757-9},

issn = {1750-2799},

year  = {2023},

date = {2023-01-01},

journal = {Nat Protoc},

volume = {18},

number = {1},

pages = {239--264},

abstract = {Cryogenic electron microscopy (cryo-EM) data represent density maps of macromolecular systems at atomic or near-atomic resolution. However, building and refining 3D atomic models by using data from cryo-EM maps is not straightforward and requires significant hands-on experience and manual intervention. We recently developed StarMap, an easy-to-use interface between the popular structural display program ChimeraX and Rosetta, a powerful molecular modeling engine. StarMap offers a general approach for refining structural models of biological macromolecules into cryo-EM density maps by combining Monte Carlo sampling with local density-guided optimization, Rosetta-based all-atom refinement and real-space B-factor calculations in a straightforward workflow. StarMap includes options for structural symmetry, local refinements and independent model validation. The overall quality of the refinement and the structure resolution is then assessed via analytical outputs, such as magnification calibration (pixel size calibration) and Fourier shell correlations. Z-scores reported by StarMap provide an easily interpretable indicator of the goodness of fit for each residue and can be plotted to evaluate structural models and improve local residue refinements, as well as to identify flexible regions and potentially functional sites in large macromolecular complexes. The protocol requires general computer skills, without the need for coding expertise, because most parts of the workflow can be operated by clicking tabs within the ChimeraX graphical user interface. Time requirements for the model refinement depend on the size and quality of the input data; however, this step can typically be completed within 1 d. The analytical parts of the workflow are completed within minutes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36108048,

title = {Hallucinating symmetric protein assemblies},

author = {B I M Wicky and L F Milles and A Courbet and R J Ragotte and J Dauparas and E Kinfu and S Tipps and R D Kibler and M Baek and F DiMaio and X Li and L Carter and A Kang and H Nguyen and A K Bera and D Baker},

doi = {10.1126/science.add1964},

issn = {1095-9203},

year  = {2022},

date = {2022-10-01},

journal = {Science},

volume = {378},

number = {6615},

pages = {56--61},

abstract = {Deep learning generative approaches provide an opportunity to broadly explore protein structure space beyond the sequences and structures of natural proteins. Here, we use deep network hallucination to generate a wide range of symmetric protein homo-oligomers given only a specification of the number of protomers and the protomer length. Crystal structures of seven designs are very similar to the computational models (median root mean square deviation: 0.6 angstroms), as are three cryo-electron microscopy structures of giant 10-nanometer rings with up to 1550 residues and  symmetry; all differ considerably from previously solved structures. Our results highlight the rich diversity of new protein structures that can be generated using deep learning and pave the way for the design of increasingly complex components for nanomachines and biomaterials.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35862514,

title = {Scaffolding protein functional sites using deep learning},

author = {Jue Wang and Sidney Lisanza and David Juergens and Doug Tischer and Joseph L Watson and Karla M Castro and Robert Ragotte and Amijai Saragovi and Lukas F Milles and Minkyung Baek and Ivan Anishchenko and Wei Yang and Derrick R Hicks and Marc Expòsit and Thomas Schlichthaerle and Jung-Ho Chun and Justas Dauparas and Nathaniel Bennett and Basile I M Wicky and Andrew Muenks and Frank DiMaio and Bruno Correia and Sergey Ovchinnikov and David Baker},

doi = {10.1126/science.abn2100},

issn = {1095-9203},

year  = {2022},

date = {2022-07-01},

journal = {Science},

volume = {377},

number = {6604},

pages = {387--394},

abstract = {The binding and catalytic functions of proteins are generally mediated by a small number of functional residues held in place by the overall protein structure. Here, we describe deep learning approaches for scaffolding such functional sites without needing to prespecify the fold or secondary structure of the scaffold. The first approach, "constrained hallucination," optimizes sequences such that their predicted structures contain the desired functional site. The second approach, "inpainting," starts from the functional site and fills in additional sequence and structure to create a viable protein scaffold in a single forward pass through a specifically trained RoseTTAFold network. We use these two methods to design candidate immunogens, receptor traps, metalloproteins, enzymes, and protein-binding proteins and validate the designs using a combination of in silico and experimental tests.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35762713,

title = {AlphaFold accurately predicts distinct conformations based on the oligomeric state of a de novo designed protein},

author = {Matthew C Cummins and Tim M Jacobs and Frank D Teets and Frank DiMaio and Ashutosh Tripathy and Brian Kuhlman},

doi = {10.1002/pro.4368},

issn = {1469-896X},

year  = {2022},

date = {2022-07-01},

journal = {Protein Sci},

volume = {31},

number = {7},

pages = {e4368},

abstract = {Using the molecular modeling program Rosetta, we designed a de novo protein, called SEWN0.1, which binds the heterotrimeric G protein Gα The design is helical, well-folded, and primarily monomeric in solution at a concentration of 10 μM. However, when we solved the crystal structure of SEWN0.1 at 1.9 Å, we observed a dimer in a conformation incompatible with binding Gα . Unintentionally, we had designed a protein that adopts alternate conformations depending on its oligomeric state. Recently, there has been tremendous progress in the field of protein structure prediction as new methods in artificial intelligence have been used to predict structures with high accuracy. We were curious if the structure prediction method AlphaFold could predict the structure of SEWN0.1 and if the prediction depended on oligomeric state. When AlphaFold was used to predict the structure of monomeric SEWN0.1, it produced a model that resembles the Rosetta design model and is compatible with binding Gα , but when used to predict the structure of a dimer, it predicted a conformation that closely resembles the SEWN0.1 crystal structure. AlphaFold's ability to predict multiple conformations for a single protein sequence should be useful for engineering protein switches.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35239409,

title = {Architecture and antigenicity of the Nipah virus attachment glycoprotein},

author = {Zhaoqian Wang and Moushimi Amaya and Amin Addetia and Ha V Dang and Gabriella Reggiano and Lianying Yan and Andrew C Hickey and Frank DiMaio and Christopher C Broder and David Veesler},

doi = {10.1126/science.abm5561},

issn = {1095-9203},

year  = {2022},

date = {2022-03-01},

journal = {Science},

volume = {375},

number = {6587},

pages = {1373--1378},

abstract = {Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness. The entry of HNVs into host cells requires the attachment (G) and fusion (F) glycoproteins, which are the main targets of antibody responses. To understand viral infection and host immunity, we determined a cryo-electron microscopy structure of the NiV G homotetrameric ectodomain in complex with the nAH1.3 broadly neutralizing antibody Fab fragment. We show that a cocktail of two nonoverlapping G-specific antibodies neutralizes NiV and HeV synergistically and limits the emergence of escape mutants. Analysis of polyclonal serum antibody responses elicited by vaccination of macaques with NiV G indicates that the receptor binding head domain is immunodominant. These results pave the way for implementing multipronged therapeutic strategies against these deadly pathogens.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35210614,

title = {Seipin forms a flexible cage at lipid droplet formation sites},

author = {Henning Arlt and Xuewu Sui and Brayden Folger and Carson Adams and Xiao Chen and Roman Remme and Fred A Hamprecht and Frank DiMaio and Maofu Liao and Joel M Goodman and Robert V Farese and Tobias C Walther},

doi = {10.1038/s41594-021-00718-y},

issn = {1545-9985},

year  = {2022},

date = {2022-03-01},

journal = {Nat Struct Mol Biol},

volume = {29},

number = {3},

pages = {194--202},

abstract = {Lipid droplets (LDs) form in the endoplasmic reticulum by phase separation of neutral lipids. This process is facilitated by the seipin protein complex, which consists of a ring of seipin monomers, with a yet unclear function. Here, we report a structure of S. cerevisiae seipin based on cryogenic-electron microscopy and structural modeling data. Seipin forms a decameric, cage-like structure with the lumenal domains forming a stable ring at the cage floor and transmembrane segments forming the cage sides and top. The transmembrane segments interact with adjacent monomers in two distinct, alternating conformations. These conformations result from changes in switch regions, located between the lumenal domains and the transmembrane segments, that are required for seipin function. Our data indicate a model for LD formation in which a closed seipin cage enables triacylglycerol phase separation and subsequently switches to an open conformation to allow LD growth and budding.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35286324,

title = {Large-scale design and refinement of stable proteins using sequence-only models},

author = {Jedediah M Singer and Scott Novotney and Devin Strickland and Hugh K Haddox and Nicholas Leiby and Gabriel J Rocklin and Cameron M Chow and Anindya Roy and Asim K Bera and Francis C Motta and Longxing Cao and Eva-Maria Strauch and Tamuka M Chidyausiku and Alex Ford and Ethan Ho and Alexander Zaitzeff and Craig O Mackenzie and Hamed Eramian and Frank DiMaio and Gevorg Grigoryan and Matthew Vaughn and Lance J Stewart and David Baker and Eric Klavins},

doi = {10.1371/journal.pone.0265020},

issn = {1932-6203},

year  = {2022},

date = {2022-01-01},

journal = {PLoS One},

volume = {17},

number = {3},

pages = {e0265020},

abstract = {Engineered proteins generally must possess a stable structure in order to achieve their designed function. Stable designs, however, are astronomically rare within the space of all possible amino acid sequences. As a consequence, many designs must be tested computationally and experimentally in order to find stable ones, which is expensive in terms of time and resources. Here we use a high-throughput, low-fidelity assay to experimentally evaluate the stability of approximately 200,000 novel proteins. These include a wide range of sequence perturbations, providing a baseline for future work in the field. We build a neural network model that predicts protein stability given only sequences of amino acids, and compare its performance to the assayed values. We also report another network model that is able to generate the amino acid sequences of novel stable proteins given requested secondary sequences. Finally, we show that the predictive model-despite weaknesses including a noisy data set-can be used to substantially increase the stability of both expert-designed and model-generated proteins.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35050657,

title = {Structural and functional insight into regulation of kinesin-1 by microtubule-associated protein MAP7},

author = {Luke S Ferro and Qianglin Fang and Lisa Eshun-Wilson and Jonathan Fernandes and Amanda Jack and Daniel P Farrell and Mert Golcuk and Teun Huijben and Katelyn Costa and Mert Gur and Frank DiMaio and Eva Nogales and Ahmet Yildiz},

doi = {10.1126/science.abf6154},

issn = {1095-9203},

year  = {2022},

date = {2022-01-01},

journal = {Science},

volume = {375},

number = {6578},

pages = {326--331},

abstract = {Microtubule (MT)-associated protein 7 (MAP7) is a required cofactor for kinesin-1-driven transport of intracellular cargoes. Using cryo-electron microscopy and single-molecule imaging, we investigated how MAP7 binds MTs and facilitates kinesin-1 motility. The MT-binding domain (MTBD) of MAP7 bound MTs as an extended α helix between the protofilament ridge and the site of lateral contact. Unexpectedly, the MTBD partially overlapped with the binding site of kinesin-1 and inhibited its motility. However, by tethering kinesin-1 to the MT, the projection domain of MAP7 prevented dissociation of the motor and facilitated its binding to available neighboring sites. The inhibitory effect of the MTBD dominated as MTs became saturated with MAP7. Our results reveal biphasic regulation of kinesin-1 by MAP7 in the context of their competitive binding to MTs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34936652,

title = {Treponema pallidum genome sequencing from six continents reveals variability in vaccine candidate genes and dominance of Nichols clade strains in Madagascar},

author = {Nicole A P Lieberman and Michelle J Lin and Hong Xie and Lasata Shrestha and Tien Nguyen and Meei-Li Huang and Austin M Haynes and Emily Romeis and Qian-Qiu Wang and Rui-Li Zhang and Cai-Xia Kou and Giulia Ciccarese and Ivano Dal Conte and Marco Cusini and Francesco Drago and Shu-Ichi Nakayama and Kenichi Lee and Makoto Ohnishi and Kelika A Konda and Silver K Vargas and Maria Eguiluz and Carlos F Caceres and Jeffrey D Klausner and Oriol Mitjà and Anne Rompalo and Fiona Mulcahy and Edward W Hook and Sheila A Lukehart and Amanda M Casto and Pavitra Roychoudhury and Frank DiMaio and Lorenzo Giacani and Alexander L Greninger},

doi = {10.1371/journal.pntd.0010063},

issn = {1935-2735},

year  = {2021},

date = {2021-12-01},

journal = {PLoS Negl Trop Dis},

volume = {15},

number = {12},

pages = {e0010063},

abstract = {In spite of its immutable susceptibility to penicillin, Treponema pallidum (T. pallidum) subsp. pallidum continues to cause millions of cases of syphilis each year worldwide, resulting in significant morbidity and mortality and underscoring the urgency of developing an effective vaccine to curtail the spread of the infection. Several technical challenges, including absence of an in vitro culture system until very recently, have hampered efforts to catalog the diversity of strains collected worldwide. Here, we provide near-complete genomes from 196 T. pallidum strains-including 191 T. pallidum subsp. pallidum-sequenced directly from patient samples collected from 8 countries and 6 continents. Maximum likelihood phylogeny revealed that samples from most sites were predominantly SS14 clade. However, 99% (84/85) of the samples from Madagascar formed two of the five distinct Nichols subclades. Although recombination was uncommon in the evolution of modern circulating strains, we found multiple putative recombination events between T. pallidum subsp. pallidum and subsp. endemicum, shaping the genomes of several subclades. Temporal analysis dated the most recent common ancestor of Nichols and SS14 clades to 1717 (95% HPD: 1543-1869), in agreement with other recent studies. Rates of SNP accumulation varied significantly among subclades, particularly among different Nichols subclades, and was associated in the Nichols A subclade with a C394F substitution in TP0380, a ERCC3-like DNA repair helicase. Our data highlight the role played by variation in genes encoding putative surface-exposed outer membrane proteins in defining separate lineages, and provide a critical resource for the design of broadly protective syphilis vaccines targeting surface antigens.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34853475,

title = {De novo protein design by deep network hallucination},

author = {Ivan Anishchenko and Samuel J Pellock and Tamuka M Chidyausiku and Theresa A Ramelot and Sergey Ovchinnikov and Jingzhou Hao and Khushboo Bafna and Christoffer Norn and Alex Kang and Asim K Bera and Frank DiMaio and Lauren Carter and Cameron M Chow and Gaetano T Montelione and David Baker},

doi = {10.1038/s41586-021-04184-w},

issn = {1476-4687},

year  = {2021},

date = {2021-12-01},

journal = {Nature},

volume = {600},

number = {7889},

pages = {547--552},

abstract = {There has been considerable recent progress in protein structure prediction using deep neural networks to predict inter-residue distances from amino acid sequences. Here we investigate whether the information captured by such networks is sufficiently rich to generate new folded proteins with sequences unrelated to those of the naturally occurring proteins used in training the models. We generate random amino acid sequences, and input them into the trRosetta structure prediction network to predict starting residue-residue distance maps, which, as expected, are quite featureless. We then carry out Monte Carlo sampling in amino acid sequence space, optimizing the contrast (Kullback-Leibler divergence) between the inter-residue distance distributions predicted by the network and background distributions averaged over all proteins. Optimization from different random starting points resulted in novel proteins spanning a wide range of sequences and predicted structures. We obtained synthetic genes encoding 129 of the network-'hallucinated' sequences, and expressed and purified the proteins in Escherichia coli; 27 of the proteins yielded monodisperse species with circular dichroism spectra consistent with the hallucinated structures. We determined the three-dimensional structures of three of the hallucinated proteins, two by X-ray crystallography and one by NMR, and these closely matched the hallucinated models. Thus, deep networks trained to predict native protein structures from their sequences can be inverted to design new proteins, and such networks and methods should contribute alongside traditional physics-based models to the de novo design of proteins with new functions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34845212,

title = {Ensuring scientific reproducibility in bio-macromolecular modeling via extensive, automated benchmarks},

author = {Julia Koehler Leman and Sergey Lyskov and Steven M Lewis and Jared Adolf-Bryfogle and Rebecca F Alford and Kyle Barlow and Ziv Ben-Aharon and Daniel Farrell and Jason Fell and William A Hansen and Ameya Harmalkar and Jeliazko Jeliazkov and Georg Kuenze and Justyna D Krys and Ajasja Ljubetič and Amanda L Loshbaugh and Jack Maguire and Rocco Moretti and Vikram Khipple Mulligan and Morgan L Nance and Phuong T Nguyen and Shane Ó Conchúir and Shourya S Roy Burman and Rituparna Samanta and Shannon T Smith and Frank Teets and Johanna K S Tiemann and Andrew Watkins and Hope Woods and Brahm J Yachnin and Christopher D Bahl and Chris Bailey-Kellogg and David Baker and Rhiju Das and Frank DiMaio and Sagar D Khare and Tanja Kortemme and Jason W Labonte and Kresten Lindorff-Larsen and Jens Meiler and William Schief and Ora Schueler-Furman and Justin B Siegel and Amelie Stein and Vladimir Yarov-Yarovoy and Brian Kuhlman and Andrew Leaver-Fay and Dominik Gront and Jeffrey J Gray and Richard Bonneau},

doi = {10.1038/s41467-021-27222-7},

issn = {2041-1723},

year  = {2021},

date = {2021-11-01},

journal = {Nat Commun},

volume = {12},

number = {1},

pages = {6947},

abstract = {Each year vast international resources are wasted on irreproducible research. The scientific community has been slow to adopt standard software engineering practices, despite the increases in high-dimensional data, complexities of workflows, and computational environments. Here we show how scientific software applications can be created in a reproducible manner when simple design goals for reproducibility are met. We describe the implementation of a test server framework and 40 scientific benchmarks, covering numerous applications in Rosetta bio-macromolecular modeling. High performance computing cluster integration allows these benchmarks to run continuously and automatically. Detailed protocol captures are useful for developers and users of Rosetta and other macromolecular modeling tools. The framework and design concepts presented here are valuable for developers and users of any type of scientific software and for the scientific community to create reproducible methods. Specific examples highlight the utility of this framework, and the comprehensive documentation illustrates the ease of adding new tests in a matter of hours.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34734801,

title = {Cryo-EM structures of CTP synthase filaments reveal mechanism of pH-sensitive assembly during budding yeast starvation},

author = {Jesse M Hansen and Avital Horowitz and Eric M Lynch and Daniel P Farrell and Joel Quispe and Frank DiMaio and Justin M Kollman},

doi = {10.7554/eLife.73368},

issn = {2050-084X},

year  = {2021},

date = {2021-11-01},

journal = {Elife},

volume = {10},

abstract = {Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or pH-insensitive mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34328683,

title = {Discovery and Characterization of Spike N-Terminal Domain-Binding Aptamers for Rapid SARS-CoV-2 Detection},

author = {Nataly Kacherovsky and Lucy F Yang and Ha V Dang and Emmeline L Cheng and Ian I Cardle and Alexandra C Walls and Matthew McCallum and Drew L Sellers and Frank DiMaio and Stephen J Salipante and Davide Corti and David Veesler and Suzie H Pun},

doi = {10.1002/anie.202107730},

issn = {1521-3773},

year  = {2021},

date = {2021-09-01},

journal = {Angew Chem Int Ed Engl},

volume = {60},

number = {39},

pages = {21211--21215},

abstract = {The coronavirus disease 2019 (COVID-19) pandemic has devastated families and disrupted healthcare, economies and societies across the globe. Molecular recognition agents that are specific for distinct viral proteins are critical components for rapid diagnostics and targeted therapeutics. In this work, we demonstrate the selection of novel DNA aptamers that bind to the SARS-CoV-2 spike glycoprotein with high specificity and affinity (<80 nM). Through binding assays and high resolution cryo-EM, we demonstrate that SNAP1 (SARS-CoV-2 spike protein N-terminal domain-binding aptamer 1) binds to the S N-terminal domain. We applied SNAP1 in lateral flow assays (LFAs) and ELISAs to detect UV-inactivated SARS-CoV-2 at concentrations as low as 5×10  copies mL . SNAP1 is therefore a promising molecular tool for SARS-CoV-2 diagnostics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34452907,

title = {Structure of the phosphoinositide 3-kinase (PI3K) p110γ-p101 complex reveals molecular mechanism of GPCR activation},

author = {Manoj K Rathinaswamy and Udit Dalwadi and Kaelin D Fleming and Carson Adams and Jordan T B Stariha and Els Pardon and Minkyung Baek and Oscar Vadas and Frank DiMaio and Jan Steyaert and Scott D Hansen and Calvin K Yip and John E Burke},

doi = {10.1126/sciadv.abj4282},

issn = {2375-2548},

year  = {2021},

date = {2021-08-01},

journal = {Sci Adv},

volume = {7},

number = {35},

abstract = {The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein-coupled receptors. Here, we report the cryo-electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gβγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gβγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ-adaptor binding domain interfaces enhanced Gβγ activation. A nanobody that specifically binds to the p101-Gβγ interface blocks activation, providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34282049,

title = {Accurate prediction of protein structures and interactions using a three-track neural network},

author = {Minkyung Baek and Frank DiMaio and Ivan Anishchenko and Justas Dauparas and Sergey Ovchinnikov and Gyu Rie Lee and Jue Wang and Qian Cong and Lisa N Kinch and R Dustin Schaeffer and Claudia Millán and Hahnbeom Park and Carson Adams and Caleb R Glassman and Andy DeGiovanni and Jose H Pereira and Andria V Rodrigues and Alberdina A van Dijk and Ana C Ebrecht and Diederik J Opperman and Theo Sagmeister and Christoph Buhlheller and Tea Pavkov-Keller and Manoj K Rathinaswamy and Udit Dalwadi and Calvin K Yip and John E Burke and K Christopher Garcia and Nick V Grishin and Paul D Adams and Randy J Read and David Baker},

doi = {10.1126/science.abj8754},

issn = {1095-9203},

year  = {2021},

date = {2021-08-01},

journal = {Science},

volume = {373},

number = {6557},

pages = {871--876},

abstract = {DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34042235,

title = {Cryo-electron Microscopy Imaging of Alzheimer's Amyloid-beta 42 Oligomer Displayed on a Functionally and Structurally Relevant Scaffold},

author = {Jinming Wu and Thorsten B Blum and Daniel P Farrell and Frank DiMaio and Jan Pieter Abrahams and Jinghui Luo},

doi = {10.1002/anie.202104497},

issn = {1521-3773},

year  = {2021},

date = {2021-08-01},

journal = {Angew Chem Int Ed Engl},

volume = {60},

number = {34},

pages = {18680--18687},

abstract = {Amyloid-β peptide (Aβ) oligomers are pathogenic species of amyloid aggregates in Alzheimer's disease. Like certain protein toxins, Aβ oligomers permeabilize cellular membranes, presumably through a pore formation mechanism. Owing to their structural and stoichiometric heterogeneity, the structure of these pores remains to be characterized. We studied a functional Aβ42-pore equivalent, created by fusing Aβ42 to the oligomerizing, soluble domain of the α-hemolysin (αHL) toxin. Our data reveal Aβ42-αHL oligomers to share major structural, functional, and biological properties with wild-type Aβ42-pores. Single-particle cryo-EM analysis of Aβ42-αHL oligomers (with an overall 3.3 Å resolution) reveals the Aβ42-pore region to be intrinsically flexible. The Aβ42-αHL oligomers will allow many of the features of the wild-type amyloid oligomers to be studied that cannot be otherwise, and may be a highly specific antigen for the development of immuno-base diagnostics and therapies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34272285,

title = {Computational design of a synthetic PD-1 agonist},

author = {Cassie M Bryan and Gabriel J Rocklin and Matthew J Bick and Alex Ford and Sonia Majri-Morrison and Ashley V Kroll and Chad J Miller and Lauren Carter and Inna Goreshnik and Alex Kang and Frank DiMaio and Kristin V Tarbell and David Baker},

doi = {10.1073/pnas.2102164118},

issn = {1091-6490},

year  = {2021},

date = {2021-07-01},

journal = {Proc Natl Acad Sci U S A},

volume = {118},

number = {29},

abstract = {Programmed cell death protein-1 (PD-1) expressed on activated T cells inhibits T cell function and proliferation to prevent an excessive immune response, and disease can result if this delicate balance is shifted in either direction. Tumor cells often take advantage of this pathway by overexpressing the PD-1 ligand PD-L1 to evade destruction by the immune system. Alternatively, if there is a decrease in function of the PD-1 pathway, unchecked activation of the immune system and autoimmunity can result. Using a combination of computation and experiment, we designed a hyperstable 40-residue miniprotein, PD-MP1, that specifically binds murine and human PD-1 at the PD-L1 interface with a K of ∼100 nM. The apo crystal structure shows that the binder folds as designed with a backbone RMSD of 1.3 Å to the design model. Trimerization of PD-MP1 resulted in a PD-1 agonist that strongly inhibits murine T cell activation. This small, hyperstable PD-1 binding protein was computationally designed with an all-beta interface, and the trimeric agonist could contribute to treatments for autoimmune and inflammatory diseases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34031912,

title = {Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters},

author = {Gamma Chi and Rebecca Ebenhoch and Henry Man and Haiping Tang and Laurence E Tremblay and Gabriella Reggiano and Xingyu Qiu and Tina Bohstedt and Idlir Liko and Fernando G Almeida and Alexandre P Garneau and Dong Wang and Gavin McKinley and Christophe P Moreau and Kiran D Bountra and Patrizia Abrusci and Shubhashish M M Mukhopadhyay and Alejandra Fernandez-Cid and Samira Slimani and Julie L Lavoie and Nicola A Burgess-Brown and Ben Tehan and Frank DiMaio and Ali Jazayeri and Paul Isenring and Carol V Robinson and Katharina L Dürr},

doi = {10.15252/embj.2020107294},

issn = {1460-2075},

year  = {2021},

date = {2021-07-01},

journal = {EMBO J},

volume = {40},

number = {14},

pages = {e107294},

abstract = {Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34188171,

title = {Structure and lipid dynamics in the maintenance of lipid asymmetry inner membrane complex of A. baumannii},

author = {Daniel Mann and Junping Fan and Kamolrat Somboon and Daniel P Farrell and Andrew Muenks and Svetomir B Tzokov and Frank DiMaio and Syma Khalid and Samuel I Miller and Julien R C Bergeron},

doi = {10.1038/s42003-021-02318-4},

issn = {2399-3642},

year  = {2021},

date = {2021-06-01},

journal = {Commun Biol},

volume = {4},

number = {1},

pages = {817},

abstract = {Multi-resistant bacteria are a major threat in modern medicine. The gram-negative coccobacillus Acinetobacter baumannii currently leads the WHO list of pathogens in critical need for new therapeutic development. The maintenance of lipid asymmetry (MLA) protein complex is one of the core machineries that transport lipids from/to the outer membrane in gram-negative bacteria. It also contributes to broad-range antibiotic resistance in several pathogens, most prominently in A. baumannii. Nonetheless, the molecular details of its role in lipid transport has remained largely elusive. Here, we report the cryo-EM maps of the core MLA complex, MlaBDEF, from the pathogen A. baumannii, in the apo-, ATP- and ADP-bound states, revealing multiple lipid binding sites in the cytosolic and periplasmic side of the complex. Molecular dynamics simulations suggest their potential trajectory across the membrane. Collectively with the recently-reported structures of the E. coli orthologue, this data also allows us to propose a molecular mechanism of lipid transport by the MLA system.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33990563,

title = {Electromechanical coupling mechanism for activation and inactivation of an HCN channel},

author = {Gucan Dai and Teresa K Aman and Frank DiMaio and William N Zagotta},

doi = {10.1038/s41467-021-23062-7},

issn = {2041-1723},

year  = {2021},

date = {2021-05-01},

journal = {Nat Commun},

volume = {12},

number = {1},

pages = {2802},

abstract = {Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET, patch-clamp fluorometry and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removing cAMP produced a largely rigid-body rotation of the C-linker relative to the transmembrane domain, bringing the A' helix of the C-linker in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not the presence of cAMP. These results suggest that - in contrast to electromechanical coupling for channel activation - the A' helix serves to couple the S4-helix movement for channel inactivation, which is likely a conserved mechanism for CNBD-family channels.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33249652,

title = {Perturbing the energy landscape for improved packing during computational protein design},

author = {Jack B Maguire and Hugh K Haddox and Devin Strickland and Samer F Halabiya and Brian Coventry and Jermel R Griffin and Surya V S R K Pulavarti and Matthew Cummins and David F Thieker and Eric Klavins and Thomas Szyperski and Frank DiMaio and David Baker and Brian Kuhlman},

doi = {10.1002/prot.26030},

issn = {1097-0134},

year  = {2021},

date = {2021-04-01},

journal = {Proteins},

volume = {89},

number = {4},

pages = {436--449},

abstract = {The FastDesign protocol in the molecular modeling program Rosetta iterates between sequence optimization and structure refinement to stabilize de novo designed protein structures and complexes. FastDesign has been used previously to design novel protein folds and assemblies with important applications in research and medicine. To promote sampling of alternative conformations and sequences, FastDesign includes stages where the energy landscape is smoothened by reducing repulsive forces. Here, we discover that this process disfavors larger amino acids in the protein core because the protein compresses in the early stages of refinement. By testing alternative ramping strategies for the repulsive weight, we arrive at a scheme that produces lower energy designs with more native-like sequence composition in the protein core. We further validate the protocol by designing and experimentally characterizing over 4000 proteins and show that the new protocol produces higher stability proteins.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33589814,

title = {BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1},

author = {Samuel R Witus and Anika L Burrell and Daniel P Farrell and Jianming Kang and Meiling Wang and Jesse M Hansen and Alex Pravat and Lisa M Tuttle and Mikaela D Stewart and Peter S Brzovic and Champak Chatterjee and Weixing Zhao and Frank DiMaio and Justin M Kollman and Rachel E Klevit},

doi = {10.1038/s41594-020-00556-4},

issn = {1545-9985},

year  = {2021},

date = {2021-03-01},

journal = {Nat Struct Mol Biol},

volume = {28},

number = {3},

pages = {268--277},

abstract = {Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in individuals with cancer. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33577321,

title = {Force Field Optimization Guided by Small Molecule Crystal Lattice Data Enables Consistent Sub-Angstrom Protein-Ligand Docking},

author = {Hahnbeom Park and Guangfeng Zhou and Minkyung Baek and David Baker and Frank DiMaio},

doi = {10.1021/acs.jctc.0c01184},

issn = {1549-9626},

year  = {2021},

date = {2021-03-01},

journal = {J Chem Theory Comput},

volume = {17},

number = {3},

pages = {2000--2010},

abstract = {Accurate and rapid calculation of protein-small molecule interaction free energies is critical for computational drug discovery. Because of the large chemical space spanned by drug-like molecules, classical force fields contain thousands of parameters describing atom-pair distance and torsional preferences; each parameter is typically optimized independently on simple representative molecules. Here, we describe a new approach in which small molecule force field parameters are jointly optimized guided by the rich source of information contained within thousands of available small molecule crystal structures. We optimize parameters by requiring that the experimentally determined molecular lattice arrangements have lower energy than all alternative lattice arrangements. Thousands of independent crystal lattice-prediction simulations were run on each of 1386 small molecule crystal structures, and energy function parameters of an implicit solvent energy model were optimized, so native crystal lattice arrangements had the lowest energy. The resulting energy model was implemented in Rosetta, together with a rapid genetic algorithm docking method employing grid-based scoring and receptor flexibility. The success rate of bound structure recapitulation in cross-docking on 1112 complexes was improved by more than 10% over previously published methods, with solutions within <1 Å in over half of the cases. Our results demonstrate that small molecule crystal structures are a rich source of information for guiding molecular force field development, and the improved Rosetta energy function should increase accuracy in a wide range of small molecule structure prediction and design studies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33542514,

title = {Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge},

author = {Catherine L Lawson and Andriy Kryshtafovych and Paul D Adams and Pavel V Afonine and Matthew L Baker and Benjamin A Barad and Paul Bond and Tom Burnley and Renzhi Cao and Jianlin Cheng and Grzegorz Chojnowski and Kevin Cowtan and Ken A Dill and Frank DiMaio and Daniel P Farrell and James S Fraser and Mark A Herzik and Soon Wen Hoh and Jie Hou and Li-Wei Hung and Maxim Igaev and Agnel P Joseph and Daisuke Kihara and Dilip Kumar and Sumit Mittal and Bohdan Monastyrskyy and Mateusz Olek and Colin M Palmer and Ardan Patwardhan and Alberto Perez and Jonas Pfab and Grigore D Pintilie and Jane S Richardson and Peter B Rosenthal and Daipayan Sarkar and Luisa U Schäfer and Michael F Schmid and Gunnar F Schröder and Mrinal Shekhar and Dong Si and Abishek Singharoy and Genki Terashi and Thomas C Terwilliger and Andrea Vaiana and Liguo Wang and Zhe Wang and Stephanie A Wankowicz and Christopher J Williams and Martyn Winn and Tianqi Wu and Xiaodi Yu and Kaiming Zhang and Helen M Berman and Wah Chiu},

doi = {10.1038/s41592-020-01051-w},

issn = {1548-7105},

year  = {2021},

date = {2021-02-01},

journal = {Nat Methods},

volume = {18},

number = {2},

pages = {156--164},

abstract = {This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33328352,

title = {Suppressor Mutations in Type II Secretion Mutants of Vibrio cholerae: Inactivation of the VesC Protease},

author = {Chelsea S Rule and Young-Jun Park and Jaclyn R Delarosa and Stewart Turley and Wim G J Hol and Sarah McColm and Colby Gura and Frank DiMaio and Konstantin V Korotkov and Maria Sandkvist},

doi = {10.1128/mSphere.01125-20},

issn = {2379-5042},

year  = {2020},

date = {2020-12-01},

journal = {mSphere},

volume = {5},

number = {6},

abstract = {The type II secretion system (T2SS) is a conserved transport pathway responsible for the secretion of a range of virulence factors by many pathogens, including  Disruption of the T2SS genes in  results in loss of secretion, changes in cell envelope function, and growth defects. While T2SS mutants are viable, high-throughput genomic analyses have listed these genes among essential genes. To investigate whether secondary mutations arise as a consequence of T2SS inactivation, we sequenced the genomes of six  T2SS mutants with deletions or insertions in either the , , or  genes and identified secondary mutations in all mutants. Two of the six T2SS mutants contain distinct mutations in the gene encoding the T2SS-secreted protease VesC. Other mutations were found in genes coding for  cell envelope proteins. Subsequent sequence analysis of the  gene in 92 additional T2SS mutant isolates identified another 19 unique mutations including insertions or deletions, sequence duplications, and single-nucleotide changes resulting in amino acid substitutions in the VesC protein. Analysis of VesC variants and the X-ray crystallographic structure of wild-type VesC suggested that all mutations lead to loss of VesC production and/or function. One possible mechanism by which  T2SS mutagenesis can be tolerated is through selection of -inactivating mutations, which may, in part, suppress cell envelope damage, establishing permissive conditions for the disruption of the T2SS. Other mutations may have been acquired in genes encoding essential cell envelope proteins to prevent proteolysis by VesC. Genome-wide transposon mutagenesis has identified the genes encoding the T2SS in  as essential for viability, but the reason for this is unclear. Mutants with deletions or insertions in these genes can be isolated, suggesting that they have acquired secondary mutations that suppress their growth defect. Through whole-genome sequencing and phenotypic analysis of T2SS mutants, we show that one means by which the growth defect can be suppressed is through mutations in the gene encoding the T2SS substrate VesC. VesC homologues are present in other  species and close relatives, and this may be why inactivation of the T2SS in species such as ,  sp. strain 60, and  also results in a pleiotropic effect on their outer membrane assembly and integrity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33182997,

title = {Crystal Structure and Mechanistic Molecular Modeling Studies of  Diterpene Cyclase Rv3377c},

author = {Yue Zhang and Lisa M Prach and Terrence E O'Brien and Frank DiMaio and Daniil M Prigozhin and Jacob E Corn and Tom Alber and Justin B Siegel and Dean J Tantillo},

doi = {10.1021/acs.biochem.0c00762},

issn = {1520-4995},

year  = {2020},

date = {2020-12-01},

journal = {Biochemistry},

volume = {59},

number = {47},

pages = {4507--4515},

abstract = {Terpenes make up the largest class of natural products, with extensive chemical and structural diversity. Diterpenes, mostly isolated from plants and rarely prokaryotes, exhibit a variety of important biological activities and valuable applications, including providing antitumor and antibiotic pharmaceuticals. These natural products are constructed by terpene synthases, a class of enzymes that catalyze one of the most complex chemical reactions in biology: converting simple acyclic oligo-isoprenyl diphosphate substrates to complex polycyclic products via carbocation intermediates. Here we obtained the second ever crystal structure of a class II diterpene synthase from bacteria, tuberculosinol pyrophosphate synthase (i.e., Halimadienyl diphosphate synthase, MtHPS, or Rv3377c) from  (). This enzyme transforms ()-geranylgeranyl diphosphate into tuberculosinol pyrophosphate (Halimadienyl diphosphate). Rv3377c is part of the  diterpene pathway along with Rv3378c, which converts tuberculosinol pyrophosphate to 1-tuberculosinyl adenosine (1-TbAd). This pathway was shown to exist only in virulent  species, but not in closely related avirulent species, and was proposed to be involved in phagolysosome maturation arrest. To gain further insight into the reaction pathway and the mechanistically relevant enzyme substrate binding orientation, electronic structure calculation and docking studies of reaction intermediates were carried out. Results reveal a plausible binding mode of the substrate that can provide the information to guide future drug design and anti-infective therapies of this biosynthetic pathway.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33058266,

title = {Computational design of mixed chirality peptide macrocycles with internal symmetry},

author = {Vikram Khipple Mulligan and Christine S Kang and Michael R Sawaya and Stephen Rettie and Xinting Li and Inna Antselovich and Timothy W Craven and Andrew M Watkins and Jason W Labonte and Frank DiMaio and Todd O Yeates and David Baker},

doi = {10.1002/pro.3974},

issn = {1469-896X},

year  = {2020},

date = {2020-12-01},

journal = {Protein Sci},

volume = {29},

number = {12},

pages = {2433--2445},

abstract = {Cyclic symmetry is frequent in protein and peptide homo-oligomers, but extremely rare within a single chain, as it is not compatible with free N- and C-termini. Here we describe the computational design of mixed-chirality peptide macrocycles with rigid structures that feature internal cyclic symmetries or improper rotational symmetries inaccessible to natural proteins. Crystal structures of three C2- and C3-symmetric macrocycles, and of six diverse S2-symmetric macrocycles, match the computationally-designed models with backbone heavy-atom RMSD values of 1 Å or better. Crystal structures of an S4-symmetric macrocycle (consisting of a sequence and structure segment mirrored at each of three successive repeats) designed to bind zinc reveal a large-scale zinc-driven conformational change from an S4-symmetric apo-state to a nearly inverted S4-symmetric holo-state almost identical to the design model. These symmetric structures provide promising starting points for applications ranging from design of cyclic peptide based metal organic frameworks to creation of high affinity binders of symmetric protein homo-oligomers. More generally, this work demonstrates the power of computational design for exploring symmetries and structures not found in nature, and for creating synthetic switchable systems.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33053319,

title = {A Structural Model of the Endogenous Human BAF Complex Informs Disease Mechanisms},

author = {Nazar Mashtalir and Hiroshi Suzuki and Daniel P Farrell and Akshay Sankar and Jie Luo and Martin Filipovski and Andrew R D'Avino and Roodolph St Pierre and Alfredo M Valencia and Takashi Onikubo and Robert G Roeder and Yan Han and Yuan He and Jeffrey A Ranish and Frank DiMaio and Thomas Walz and Cigall Kadoch},

doi = {10.1016/j.cell.2020.09.051},

issn = {1097-4172},

year  = {2020},

date = {2020-10-01},

journal = {Cell},

volume = {183},

number = {3},

pages = {802--817.e24},

abstract = {Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal α-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities. Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32956350,

title = {Efficient consideration of coordinated water molecules improves computational protein-protein and protein-ligand docking discrimination},

author = {Ryan E Pavlovicz and Hahnbeom Park and Frank DiMaio},

doi = {10.1371/journal.pcbi.1008103},

issn = {1553-7358},

year  = {2020},

date = {2020-09-01},

journal = {PLoS Comput Biol},

volume = {16},

number = {9},

pages = {e1008103},

abstract = {Highly coordinated water molecules are frequently an integral part of protein-protein and protein-ligand interfaces. We introduce an updated energy model that efficiently captures the energetic effects of these ordered water molecules on the surfaces of proteins. A two-stage method is developed in which polar groups arranged in geometries suitable for water placement are first identified, then a modified Monte Carlo simulation allows highly coordinated waters to be placed on the surface of a protein while simultaneously sampling amino acid side chain orientations. This "semi-explicit" water model is implemented in Rosetta and is suitable for both structure prediction and protein design. We show that our new approach and energy model yield significant improvements in native structure recovery of protein-protein and protein-ligand docking discrimination tests.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32939280,

title = {Deep learning enables the atomic structure determination of the Fanconi Anemia core complex from cryoEM},

author = {Daniel P Farrell and Ivan Anishchenko and Shabih Shakeel and Anna Lauko and Lori A Passmore and David Baker and Frank DiMaio},

doi = {10.1107/S2052252520009306},

issn = {2052-2525},

year  = {2020},

date = {2020-09-01},

journal = {IUCrJ},

volume = {7},

number = {Pt 5},

pages = {881--892},

abstract = {Cryo-electron microscopy of protein complexes often leads to moderate resolution maps (4-8 Å), with visible secondary-structure elements but poorly resolved loops, making model building challenging. In the absence of high-resolution structures of homologues, only coarse-grained structural features are typically inferred from these maps, and it is often impossible to assign specific regions of density to individual protein subunits. This paper describes a new method for overcoming these difficulties that integrates predicted residue distance distributions from a deep-learned convolutional neural network, computational protein folding using , and automated EM-map-guided complex assembly. We apply this method to a 4.6 Å resolution cryoEM map of Fanconi Anemia core complex (FAcc), an E3 ubiquitin ligase required for DNA interstrand crosslink repair, which was previously challenging to interpret as it comprises 6557 residues, only 1897 of which are covered by homology models. In the published model built from this map, only 387 residues could be assigned to the specific subunits with confidence. By building and placing into density 42 deep-learning-guided models containing 4795 residues not included in the previously published structure, we are able to determine an almost-complete atomic model of FAcc, in which 5182 of the 6557 residues were placed. The resulting model is consistent with previously published biochemical data, and facilitates interpretation of disease-related mutational data. We anticipate that our approach will be broadly useful for cryoEM structure determination of large complexes containing many subunits for which there are no homologues of known structure.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32848250,

title = {Computational design of transmembrane pores},

author = {Chunfu Xu and Peilong Lu and Tamer M Gamal El-Din and Xue Y Pei and Matthew C Johnson and Atsuko Uyeda and Matthew J Bick and Qi Xu and Daohua Jiang and Hua Bai and Gabriella Reggiano and Yang Hsia and T J Brunette and Jiayi Dou and Dan Ma and Eric M Lynch and Scott E Boyken and Po-Ssu Huang and Lance Stewart and Frank DiMaio and Justin M Kollman and Ben F Luisi and Tomoaki Matsuura and William A Catterall and David Baker},

doi = {10.1038/s41586-020-2646-5},

issn = {1476-4687},

year  = {2020},

date = {2020-09-01},

journal = {Nature},

volume = {585},

number = {7823},

pages = {129--134},

abstract = {Transmembrane channels and pores have key roles in fundamental biological processes and in biotechnological applications such as DNA nanopore sequencing, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels, and there have been recent advances in de novo membrane protein design and in redesigning naturally occurring channel-containing proteins. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32839327,

title = {An enumerative algorithm for de novo design of proteins with diverse pocket structures},

author = {Benjamin Basanta and Matthew J Bick and Asim K Bera and Christoffer Norn and Cameron M Chow and Lauren P Carter and Inna Goreshnik and Frank Dimaio and David Baker},

doi = {10.1073/pnas.2005412117},

issn = {1091-6490},

year  = {2020},

date = {2020-09-01},

journal = {Proc Natl Acad Sci U S A},

volume = {117},

number = {36},

pages = {22135--22145},

abstract = {To create new enzymes and biosensors from scratch, precise control over the structure of small-molecule binding sites is of paramount importance, but systematically designing arbitrary protein pocket shapes and sizes remains an outstanding challenge. Using the NTF2-like structural superfamily as a model system, we developed an enumerative algorithm for creating a virtually unlimited number of de novo proteins supporting diverse pocket structures. The enumerative algorithm was tested and refined through feedback from two rounds of large-scale experimental testing, involving in total the assembly of synthetic genes encoding 7,896 designs and assessment of their stability on yeast cell surface, detailed biophysical characterization of 64 designs, and crystal structures of 5 designs. The refined algorithm generates proteins that remain folded at high temperatures and exhibit more pocket diversity than naturally occurring NTF2-like proteins. We expect this approach to transform the design of small-molecule sensors and enzymes by enabling the creation of binding and active site geometries much more optimal for specific design challenges than is accessible by repurposing the limited number of naturally occurring NTF2-like proteins.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32632011,

title = {Structural basis for autophagy inhibition by the human Rubicon-Rab7 complex},

author = {Hersh K Bhargava and Keisuke Tabata and Jordan M Byck and Maho Hamasaki and Daniel P Farrell and Ivan Anishchenko and Frank DiMaio and Young Jun Im and Tamotsu Yoshimori and James H Hurley},

doi = {10.1073/pnas.2008030117},

issn = {1091-6490},

year  = {2020},

date = {2020-07-01},

journal = {Proc Natl Acad Sci U S A},

volume = {117},

number = {29},

pages = {17003--17010},

abstract = {Rubicon is a potent negative regulator of autophagy and a potential target for autophagy-inducing therapeutics. Rubicon-mediated inhibition of autophagy requires the interaction of the C-terminal Rubicon homology (RH) domain of Rubicon with Rab7-GTP. Here we report the 2.8-Å crystal structure of the Rubicon RH domain in complex with Rab7-GTP. Our structure reveals a fold for the RH domain built around four zinc clusters. The switch regions of Rab7 insert into pockets on the surface of the RH domain in a mode that is distinct from those of other Rab-effector complexes. Rubicon residues at the dimer interface are required for Rubicon and Rab7 to colocalize in living cells. Mutation of Rubicon RH residues in the Rab7-binding site restores efficient autophagic flux in the presence of overexpressed Rubicon, validating the Rubicon RH domain as a promising therapeutic target.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32483333,

title = {Macromolecular modeling and design in Rosetta: recent methods and frameworks},

author = {Julia Koehler Leman and Brian D Weitzner and Steven M Lewis and Jared Adolf-Bryfogle and Nawsad Alam and Rebecca F Alford and Melanie Aprahamian and David Baker and Kyle A Barlow and Patrick Barth and Benjamin Basanta and Brian J Bender and Kristin Blacklock and Jaume Bonet and Scott E Boyken and Phil Bradley and Chris Bystroff and Patrick Conway and Seth Cooper and Bruno E Correia and Brian Coventry and Rhiju Das and René M De Jong and Frank DiMaio and Lorna Dsilva and Roland Dunbrack and Alexander S Ford and Brandon Frenz and Darwin Y Fu and Caleb Geniesse and Lukasz Goldschmidt and Ragul Gowthaman and Jeffrey J Gray and Dominik Gront and Sharon Guffy and Scott Horowitz and Po-Ssu Huang and Thomas Huber and Tim M Jacobs and Jeliazko R Jeliazkov and David K Johnson and Kalli Kappel and John Karanicolas and Hamed Khakzad and Karen R Khar and Sagar D Khare and Firas Khatib and Alisa Khramushin and Indigo C King and Robert Kleffner and Brian Koepnick and Tanja Kortemme and Georg Kuenze and Brian Kuhlman and Daisuke Kuroda and Jason W Labonte and Jason K Lai and Gideon Lapidoth and Andrew Leaver-Fay and Steffen Lindert and Thomas Linsky and Nir London and Joseph H Lubin and Sergey Lyskov and Jack Maguire and Lars Malmström and Enrique Marcos and Orly Marcu and Nicholas A Marze and Jens Meiler and Rocco Moretti and Vikram Khipple Mulligan and Santrupti Nerli and Christoffer Norn and Shane Ó'Conchúir and Noah Ollikainen and Sergey Ovchinnikov and Michael S Pacella and Xingjie Pan and Hahnbeom Park and Ryan E Pavlovicz and Manasi Pethe and Brian G Pierce and Kala Bharath Pilla and Barak Raveh and P Douglas Renfrew and Shourya S Roy Burman and Aliza Rubenstein and Marion F Sauer and Andreas Scheck and William Schief and Ora Schueler-Furman and Yuval Sedan and Alexander M Sevy and Nikolaos G Sgourakis and Lei Shi and Justin B Siegel and Daniel-Adriano Silva and Shannon Smith and Yifan Song and Amelie Stein and Maria Szegedy and Frank D Teets and Summer B Thyme and Ray Yu-Ruei Wang and Andrew Watkins and Lior Zimmerman and Richard Bonneau},

doi = {10.1038/s41592-020-0848-2},

issn = {1548-7105},

year  = {2020},

date = {2020-07-01},

journal = {Nat Methods},

volume = {17},

number = {7},

pages = {665--680},

abstract = {The Rosetta software for macromolecular modeling, docking and design is extensively used in laboratories worldwide. During two decades of development by a community of laboratories at more than 60 institutions, Rosetta has been continuously refactored and extended. Its advantages are its performance and interoperability between broad modeling capabilities. Here we review tools developed in the last 5 years, including over 80 methods. We discuss improvements to the score function, user interfaces and usability. Rosetta is available at http://www.rosettacommons.org.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32587243,

title = {The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation},

author = {Natalie S Al-Otaibi and Aidan J Taylor and Daniel P Farrell and Svetomir B Tzokov and Frank DiMaio and David J Kelly and Julien R C Bergeron},

doi = {10.1038/s41467-020-16981-4},

issn = {2041-1723},

year  = {2020},

date = {2020-06-01},

journal = {Nat Commun},

volume = {11},

number = {1},

pages = {3210},

abstract = {The bacterial flagellum is a remarkable molecular motor, whose primary function in bacteria is to facilitate motility through the rotation of a filament protruding from the bacterial cell. A cap complex, consisting of an oligomer of the protein FliD, is localized at the tip of the flagellum, and is essential for filament assembly, as well as adherence to surfaces in some bacteria. However, the structure of the intact cap complex, and the molecular basis for its interaction with the filament, remains elusive. Here we report the cryo-EM structure of the Campylobacter jejuni cap complex, which reveals that FliD is pentameric, with the N-terminal region of the protomer forming an extensive set of contacts across several subunits, that contribute to FliD oligomerization. We also demonstrate that the native C. jejuni flagellum filament is 11-stranded, contrary to a previously published cryo-EM structure, and propose a molecular model for the filament-cap interaction.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32358188,

title = {Allosteric conformational change of a cyclic nucleotide-gated ion channel revealed by DEER spectroscopy},

author = {Eric G B Evans and Jacob L W Morgan and Frank DiMaio and William N Zagotta and Stefan Stoll},

doi = {10.1073/pnas.1916375117},

issn = {1091-6490},

year  = {2020},

date = {2020-05-01},

journal = {Proc Natl Acad Sci U S A},

volume = {117},

number = {20},

pages = {10839--10847},

abstract = {Cyclic nucleotide-gated (CNG) ion channels are essential components of mammalian visual and olfactory signal transduction. CNG channels open upon direct binding of cyclic nucleotides (cAMP and/or cGMP), but the allosteric mechanism by which this occurs is incompletely understood. Here, we employed double electron-electron resonance (DEER) spectroscopy to measure intersubunit distance distributions in SthK, a bacterial CNG channel from  Spin labels were introduced into the SthK C-linker, a domain that is essential for coupling cyclic nucleotide binding to channel opening. DEER revealed an agonist-dependent conformational change in which residues of the B'-helix displayed outward movement with respect to the symmetry axis of the channel in the presence of the full agonist cAMP, but not with the partial agonist cGMP. This conformational rearrangement was observed both in detergent-solubilized SthK and in channels reconstituted into lipid nanodiscs. In addition to outward movement of the B'-helix, DEER-constrained Rosetta structural models suggest that channel activation involves upward translation of the cytoplasmic domain and formation of state-dependent interactions between the C-linker and the transmembrane domain. Our results demonstrate a previously unrecognized structural transition in a CNG channel and suggest key interactions that may be responsible for allosteric gating in these channels.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32108112,

title = {Polymerization in the actin ATPase clan regulates hexokinase activity in yeast},

author = {Patrick R Stoddard and Eric M Lynch and Daniel P Farrell and Annie M Dosey and Frank DiMaio and Tom A Williams and Justin M Kollman and Andrew W Murray and Ethan C Garner},

doi = {10.1126/science.aay5359},

issn = {1095-9203},

year  = {2020},

date = {2020-02-01},

journal = {Science},

volume = {367},

number = {6481},

pages = {1039--1042},

abstract = {The actin fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, such as the carbohydrate kinases, do not polymerize. We found that Glk1, a  glucokinase, forms two-stranded filaments with ultrastructure that is distinct from that of cytoskeletal polymers. In cells, Glk1 polymerized upon sugar addition and depolymerized upon sugar withdrawal. Polymerization inhibits enzymatic activity; the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation that eliminated Glk1 polymerization alleviated concentration-dependent enzyme inhibition. Yeast containing nonpolymerizing Glk1 were less fit when growing on sugars and more likely to die when refed glucose. Glk1 polymerization arose independently from other actin-related filaments and may allow yeast to rapidly modulate glucokinase activity as nutrient availability changes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33134287,

title = {Prediction of Protein Mutational Free Energy: Benchmark and Sampling Improvements Increase Classification Accuracy},

author = {Brandon Frenz and Steven M Lewis and Indigo King and Frank DiMaio and Hahnbeom Park and Yifan Song},

doi = {10.3389/fbioe.2020.558247},

issn = {2296-4185},

year  = {2020},

date = {2020-01-01},

journal = {Front Bioeng Biotechnol},

volume = {8},

pages = {558247},

abstract = {Software to predict the change in protein stability upon point mutation is a valuable tool for a number of biotechnological and scientific problems. To facilitate the development of such software and provide easy access to the available experimental data, the ProTherm database was created. Biases in the methods and types of information collected has led to disparity in the types of mutations for which experimental data is available. For example, mutations to alanine are hugely overrepresented whereas those involving charged residues, especially from one charged residue to another, are underrepresented. ProTherm subsets created as benchmark sets that do not account for this often underrepresent tense certain mutational types. This issue introduces systematic biases into previously published protocols' ability to accurately predict the change in folding energy on these classes of mutations. To resolve this issue, we have generated a new benchmark set with these problems corrected. We have then used the benchmark set to test a number of improvements to the point mutation energetics tools in the Rosetta software suite.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31792450,

title = {Structures of MERS-CoV spike glycoprotein in complex with sialoside attachment receptors},

author = {Young-Jun Park and Alexandra C Walls and Zhaoqian Wang and Maximillian M Sauer and Wentao Li and M Alejandra Tortorici and Berend-Jan Bosch and Frank DiMaio and David Veesler},

doi = {10.1038/s41594-019-0334-7},

issn = {1545-9985},

year  = {2019},

date = {2019-12-01},

journal = {Nat Struct Mol Biol},

volume = {26},

number = {12},

pages = {1151--1157},

abstract = {The Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and often lethal respiratory illness in humans, and no vaccines or specific treatments are available. Infections are initiated via binding of the MERS-CoV spike (S) glycoprotein to sialosides and dipeptidyl-peptidase 4 (the attachment and entry receptors, respectively). To understand MERS-CoV engagement of sialylated receptors, we determined the cryo-EM structures of S in complex with 5-N-acetyl neuraminic acid, 5-N-glycolyl neuraminic acid, sialyl-Lewis, α2,3-sialyl-N-acetyl-lactosamine and α2,6-sialyl-N-acetyl-lactosamine at 2.7-3.0 Å resolution. We show that recognition occurs via a conserved groove that is essential for MERS-CoV S-mediated attachment to sialosides and entry into human airway epithelial cells. Our data illuminate MERS-CoV S sialoside specificity and suggest that selectivity for α2,3-linked over α2,6-linked receptors results from enhanced interactions with the former class of oligosaccharides. This study provides a structural framework explaining MERS-CoV attachment to sialoside receptors and identifies a site of potential vulnerability to inhibitors of viral entry.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31672968,

title = {Phase separation of Polo-like kinase 4 by autoactivation and clustering drives centriole biogenesis},

author = {Jung-Eun Park and Liang Zhang and Jeong Kyu Bang and Thorkell Andresson and Frank DiMaio and Kyung S Lee},

doi = {10.1038/s41467-019-12619-2},

issn = {2041-1723},

year  = {2019},

date = {2019-10-01},

journal = {Nat Commun},

volume = {10},

number = {1},

pages = {4959},

abstract = {Tight control of centriole duplication is critical for normal chromosome segregation and the maintenance of genomic stability. Polo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. How Plk4 dynamically promotes its symmetry-breaking relocalization and achieves its procentriole-assembly state remains unknown. Here we show that Plk4 is a unique kinase that utilizes its autophosphorylated noncatalytic cryptic polo-box (CPB) to phase separate and generate a nanoscale spherical condensate. Analyses of the crystal structure of a phospho-mimicking, condensation-proficient CPB mutant reveal that a disordered loop at the CPB PB2-tip region is critically required for Plk4 to generate condensates and induce procentriole assembly. CPB phosphorylation also promotes Plk4's dissociation from the Cep152 tether while binding to downstream STIL, thus allowing Plk4 condensate to serve as an assembling body for centriole biogenesis. This study uncovers the mechanism underlying Plk4 activation and may offer strategies for anti-Plk4 intervention against genomic instability and cancer.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31303482,

title = {The Molecular Architecture of Native BBSome Obtained by an Integrated Structural Approach},

author = {Hui-Ting Chou and Luise Apelt and Daniel P Farrell and Susan Roehl White and Jonathan Woodsmith and Vladimir Svetlov and Jaclyn S Goldstein and Andrew R Nager and Zixuan Li and Jean Muller and Hélène Dollfus and Evgeny Nudler and Ulrich Stelzl and Frank DiMaio and Maxence V Nachury and Thomas Walz},

doi = {10.1016/j.str.2019.06.006},

issn = {1878-4186},

year  = {2019},

date = {2019-09-01},

journal = {Structure},

volume = {27},

number = {9},

pages = {1384--1394.e4},

abstract = {The unique membrane composition of cilia is maintained by a diffusion barrier at the transition zone that is breached when the BBSome escorts signaling receptors out of cilia. Understanding how the BBSome removes proteins from cilia has been hampered by a lack of structural information. Here, we present a nearly complete Cα model of BBSome purified from cow retina. The model is based on a single-particle cryo-electron microscopy density map at 4.9-Å resolution that was interpreted with the help of comprehensive Rosetta-based structural modeling constrained by crosslinking mass spectrometry data. We find that BBSome subunits have a very high degree of interconnectivity, explaining the obligate nature of the complex. Furthermore, like other coat adaptors, the BBSome exists in an autoinhibited state in solution and must thus undergo a conformational change upon recruitment to membranes by the small GTPase ARL6/BBS3. Our model provides the first detailed view of the machinery enabling ciliary exit.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31200019,

title = {3.1 Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids},

author = {Indrajit Lahiri and Jun Xu and Bong Gyoon Han and Juntaek Oh and Dong Wang and Frank DiMaio and Andres E Leschziner},

doi = {10.1016/j.jsb.2019.06.004},

issn = {1095-8657},

year  = {2019},

date = {2019-09-01},

journal = {J Struct Biol},

volume = {207},

number = {3},

pages = {270--278},

abstract = {Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact with the air-water interface, which often leads to detrimental effects such as denaturation or adoption of preferred orientations, ultimately hindering structure determination. Randomly biotinylating the protein of interest (for example, at its primary amines) and then tethering it to a cryo-EM grid coated with two-dimensional crystals of streptavidin (acting as an affinity surface) can prevent the protein from interacting with the air-water interface. Recently, this approach was successfully used to solve a high-resolution structure of a test sample, a bacterial ribosome. However, whether this method can be used for samples where interaction with the air-water interface has been shown to be problematic remains to be determined. Here we report a 3.1 Å structure of an RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion (Pol II EC(CPD)) solved using streptavidin grids. Our previous attempt to solve this structure using conventional sample preparation methods resulted in a poor quality cryo-EM map due to Pol II EC(CPD)'s adopting a strong preferred orientation. Imaging the same sample on streptavidin grids improved the angular distribution of its view, resulting in a high-resolution structure. This work shows that streptavidin affinity grids can be used to address known challenges posed by the interaction with the air-water interface.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31285608,

title = {The HCN channel voltage sensor undergoes a large downward motion during hyperpolarization},

author = {Gucan Dai and Teresa K Aman and Frank DiMaio and William N Zagotta},

doi = {10.1038/s41594-019-0259-1},

issn = {1545-9985},

year  = {2019},

date = {2019-08-01},

journal = {Nat Struct Mol Biol},

volume = {26},

number = {8},

pages = {686--694},

abstract = {Voltage-gated ion channels (VGICs) contain positively charged residues within the S4 helix of the voltage-sensing domain (VSD) that are displaced in response to changes in transmembrane voltage, promoting conformational changes that open the pore. Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are unique among VGICs because their open probability is increased by membrane hyperpolarization rather than depolarization. Here we measured the precise movement of the S4 helix of a sea urchin HCN channel using transition metal ion fluorescence resonance energy transfer (tmFRET). We show that the S4 undergoes a substantial (~10 Å) downward movement in response to membrane hyperpolarization. Furthermore, by applying distance constraints determined from tmFRET experiments to Rosetta modeling, we reveal that the carboxy-terminal part of the S4 helix exhibits an unexpected tilting motion during hyperpolarization activation. These data provide a long-sought glimpse of the hyperpolarized state of a functioning VSD and also a framework for understanding the dynamics of reverse gating in HCN channels.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31110358,

title = {An extensively glycosylated archaeal pilus survives extreme conditions},

author = {Fengbin Wang and Virginija Cvirkaite-Krupovic and Mark A B Kreutzberger and Zhangli Su and Guilherme A P de Oliveira and Tomasz Osinski and Nicholas Sherman and Frank DiMaio and Joseph S Wall and David Prangishvili and Mart Krupovic and Edward H Egelman},

doi = {10.1038/s41564-019-0458-x},

issn = {2058-5276},

year  = {2019},

date = {2019-08-01},

journal = {Nat Microbiol},

volume = {4},

number = {8},

pages = {1401--1410},

abstract = {Pili on the surface of Sulfolobus islandicus are used for many functions, and serve as receptors for certain archaeal viruses. The cells grow optimally at pH 3 and ~80 °C, exposing these extracellular appendages to a very harsh environment. The pili, when removed from cells, resist digestion by trypsin or pepsin, and survive boiling in sodium dodecyl sulfate or 5 M guanidine hydrochloride. We used electron cryo-microscopy to determine the structure of these filaments at 4.1 Å resolution. An atomic model was built by combining the electron density map with bioinformatics without previous knowledge of the pilin sequence-an approach that should prove useful for assemblies where all of the components are not known. The atomic structure of the pilus was unusual, with almost one-third of the residues being either threonine or serine, and with many hydrophobic surface residues. While the map showed extra density consistent with glycosylation for only three residues, mass measurements suggested extensive glycosylation. We propose that this extensive glycosylation renders these filaments soluble and provides the remarkable structural stability. We also show that the overall fold of the archaeal pilin is remarkably similar to that of archaeal flagellin, establishing common evolutionary origins.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31260282,

title = {COMBINES-CID: An Efficient Method for De Novo Engineering of Highly Specific Chemically Induced Protein Dimerization Systems},

author = {Shoukai Kang and Kristian Davidsen and Luis Gomez-Castillo and Huayi Jiang and Xiaonan Fu and Zengpeng Li and Yu Liang and Molly Jahn and Mahmoud Moussa and Frank DiMaio and Liangcai Gu},

doi = {10.1021/jacs.9b03522},

issn = {1520-5126},

year  = {2019},

date = {2019-07-01},

journal = {J Am Chem Soc},

volume = {141},

number = {28},

pages = {10948--10952},

abstract = {Chemically induced dimerization (CID) systems, in which two proteins dimerize only in the presence of a small molecule ligand, offer versatile tools for small molecule sensing and actuation. However, only a handful of CID systems exist and creating one with the desired sensitivity and specificity for any given ligand is an unsolved problem. Here, we developed a binatorial ders-nabled election of CID (COMBINES-CID) method broadly applicable to different ligands. We demonstrated a proof-of-principle by generating nanobody-based heterodimerization systems induced by cannabidiol with high ligand selectivity. We applied the CID system to a sensitive sandwich enzyme-linked immunosorbent assay-like assay of cannabidiol in body fluids with a detection limit of ∼0.25 ng/mL. COMBINES-CID provides an efficient, cost-effective solution for expanding the biosensor toolkit for small molecule detection.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31168091,

title = {De novo protein design by citizen scientists},

author = {Brian Koepnick and Jeff Flatten and Tamir Husain and Alex Ford and Daniel-Adriano Silva and Matthew J Bick and Aaron Bauer and Gaohua Liu and Yojiro Ishida and Alexander Boykov and Roger D Estep and Susan Kleinfelter and Toke Nørgård-Solano and Linda Wei and Foldit Players and Gaetano T Montelione and Frank DiMaio and Zoran Popović and Firas Khatib and Seth Cooper and David Baker},

doi = {10.1038/s41586-019-1274-4},

issn = {1476-4687},

year  = {2019},

date = {2019-06-01},

journal = {Nature},

volume = {570},

number = {7761},

pages = {390--394},

abstract = {Online citizen science projects such as GalaxyZoo, Eyewire and Phylo have proven very successful for data collection, annotation and processing, but for the most part have harnessed human pattern-recognition skills rather than human creativity. An exception is the game EteRNA, in which game players learn to build new RNA structures by exploring the discrete two-dimensional space of Watson-Crick base pairing possibilities. Building new proteins, however, is a more challenging task to present in a game, as both the representation and evaluation of a protein structure are intrinsically three-dimensional. We posed the challenge of de novo protein design in the online protein-folding game Foldit. Players were presented with a fully extended peptide chain and challenged to craft a folded protein structure and an amino acid sequence encoding that structure. After many iterations of player design, analysis of the top-scoring solutions and subsequent game improvement, Foldit players can now-starting from an extended polypeptide chain-generate a diversity of protein structures and sequences that encode them in silico. One hundred forty-six Foldit player designs with sequences unrelated to naturally occurring proteins were encoded in synthetic genes; 56 were found to be expressed and soluble in Escherichia coli, and to adopt stable monomeric folded structures in solution. The diversity of these structures is unprecedented in de novo protein design, representing 20 different folds-including a new fold not observed in natural proteins. High-resolution structures were determined for four of the designs, and are nearly identical to the player models. This work makes explicit the considerable implicit knowledge that contributes to success in de novo protein design, and shows that citizen scientists can discover creative new solutions to outstanding scientific challenges such as the protein design problem.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31160557,

title = {Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase},

author = {Alexandrea N Rizo and JiaBei Lin and Stephanie N Gates and Eric Tse and Stephen M Bart and Laura M Castellano and Frank DiMaio and James Shorter and Daniel R Southworth},

doi = {10.1038/s41467-019-10150-y},

issn = {2041-1723},

year  = {2019},

date = {2019-06-01},

journal = {Nat Commun},

volume = {10},

number = {1},

pages = {2393},

abstract = {Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. Here we report cryo-EM structures of a hyperactive ClpB variant bound to the model substrate, casein in the presence of slowly hydrolysable ATPγS, which reveal the translocation mechanism. Distinct substrate-gripping interactions are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent to the topmost NBD1 contact. NBD conformations at the seam interface reveal how ATP hydrolysis-driven substrate disengagement and re-binding are precisely tuned to drive a directional, stepwise translocation cycle.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31142835,

title = {Multiple liquid crystalline geometries of highly compacted nucleic acid in a dsRNA virus},

author = {Serban L Ilca and Xiaoyu Sun and Kamel El Omari and Abhay Kotecha and Felix de Haas and Frank DiMaio and Jonathan M Grimes and David I Stuart and Minna M Poranen and Juha T Huiskonen},

doi = {10.1038/s41586-019-1229-9},

issn = {1476-4687},

year  = {2019},

date = {2019-06-01},

journal = {Nature},

volume = {570},

number = {7760},

pages = {252--256},

abstract = {Characterizing the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. Owing to the different cellular fates of DNA and RNA, the life cycles of double-stranded (ds)DNA and dsRNA viruses are dissimilar. In terms of nucleic acid packing, dsDNA viruses, which lack genome segmentation and intra-capsid transcriptional machinery, predominantly display single-spooled genome organizations. Because the release of dsRNA into the cytoplasm triggers host defence mechanisms, dsRNA viruses retain their genomes within a core particle that contains the enzymes required for RNA replication and transcription. The genomes of dsRNA viruses vary greatly in the degree of segmentation. In members of the Reoviridae family, genomes consist of 10-12 segments and exhibit a non-spooled arrangement mediated by RNA-dependent RNA polymerases. However, whether this arrangement is a general feature of dsRNA viruses remains unknown. Here, using cryo-electron microscopy to resolve the dsRNA genome structure of the tri-segmented bacteriophage ɸ6 of the Cystoviridae family, we show that dsRNA viruses can adopt a dsDNA-like single-spooled genome organization. We find that in this group of viruses, RNA-dependent RNA polymerases do not direct genome ordering, and the dsRNA can adopt multiple conformations. We build a model that encompasses 90% of the genome, and use this to quantify variation in the packing density and to characterize the different liquid crystalline geometries that are exhibited by the tightly compacted nucleic acid. Our results demonstrate that the canonical model for the packing of dsDNA can be extended to dsRNA viruses.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31050411,

title = {Self-Assembling 2D Arrays with de Novo Protein Building Blocks},

author = {Zibo Chen and Matthew C Johnson and Jiajun Chen and Matthew J Bick and Scott E Boyken and Baihan Lin and James J De Yoreo and Justin M Kollman and David Baker and Frank DiMaio},

doi = {10.1021/jacs.9b01978},

issn = {1520-5126},

year  = {2019},

date = {2019-06-01},

journal = {J Am Chem Soc},

volume = {141},

number = {22},

pages = {8891--8895},

abstract = {Modular self-assembly of biomolecules in two dimensions (2D) is straightforward with DNA but has been difficult to realize with proteins, due to the lack of modular specificity similar to Watson-Crick base pairing. Here we describe a general approach to design 2D arrays using de novo designed pseudosymmetric protein building blocks. A homodimeric helical bundle was reconnected into a monomeric building block, and the surface was redesigned in Rosetta to enable self-assembly into a 2D array in the C12 layer symmetry group. Two out of ten designed arrays assembled to micrometer scale under negative stain electron microscopy, and displayed the designed lattice geometry with assembly size up to 100 nm under atomic force microscopy. The design of 2D arrays with pseudosymmetric building blocks is an important step toward the design of programmable protein self-assembly via pseudosymmetric patterning of orthogonal binding interfaces.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30568301,

title = {Programmable design of orthogonal protein heterodimers},

author = {Zibo Chen and Scott E Boyken and Mengxuan Jia and Florian Busch and David Flores-Solis and Matthew J Bick and Peilong Lu and Zachary L VanAernum and Aniruddha Sahasrabuddhe and Robert A Langan and Sherry Bermeo and T J Brunette and Vikram Khipple Mulligan and Lauren P Carter and Frank DiMaio and Nikolaos G Sgourakis and Vicki H Wysocki and David Baker},

doi = {10.1038/s41586-018-0802-y},

issn = {1476-4687},

year  = {2019},

date = {2019-01-01},

journal = {Nature},

volume = {565},

number = {7737},

pages = {106--111},

abstract = {Specificity of interactions between two DNA strands, or between protein and DNA, is often achieved by varying bases or side chains coming off the DNA or protein backbone-for example, the bases participating in Watson-Crick pairing in the double helix, or the side chains contacting DNA in TALEN-DNA complexes. By contrast, specificity of protein-protein interactions usually involves backbone shape complementarity, which is less modular and hence harder to generalize. Coiled-coil heterodimers are an exception, but the restricted geometry of interactions across the heterodimer interface (primarily at the heptad a and d positions) limits the number of orthogonal pairs that can be created simply by varying side-chain interactions. Here we show that protein-protein interaction specificity can be achieved using extensive and modular side-chain hydrogen-bond networks. We used the Crick generating equations to produce millions of four-helix backbones with varying degrees of supercoiling around a central axis, identified those accommodating extensive hydrogen-bond networks, and used Rosetta to connect pairs of helices with short loops and to optimize the remainder of the sequence. Of 97 such designs expressed in Escherichia coli, 65 formed constitutive heterodimers, and the crystal structures of four designs were in close agreement with the computational models and confirmed the designed hydrogen-bond networks. In cells, six heterodimers were fully orthogonal, and in vitro-following mixing of 32 chains from 16 heterodimer designs, denaturation in 5 M guanidine hydrochloride and reannealing-almost all of the interactions observed by native mass spectrometry were between the designed cognate pairs. The ability to design orthogonal protein heterodimers should enable sophisticated protein-based control logic for synthetic biology, and illustrates that nature has not fully explored the possibilities for programmable biomolecular interaction modalities.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30344107,

title = {Automatically Fixing Errors in Glycoprotein Structures with Rosetta},

author = {Brandon Frenz and Sebastian Rämisch and Andrew J Borst and Alexandra C Walls and Jared Adolf-Bryfogle and William R Schief and David Veesler and Frank DiMaio},

doi = {10.1016/j.str.2018.09.006},

issn = {1878-4186},

year  = {2019},

date = {2019-01-01},

journal = {Structure},

volume = {27},

number = {1},

pages = {134--139.e3},

abstract = {Recent advances in single-particle cryo-electron microscopy (cryoEM) have resulted in determination of an increasing number of protein structures with resolved glycans. However, existing protocols for the refinement of glycoproteins at low resolution have failed to keep up with these advances. As a result, numerous deposited structures contain glycan stereochemical errors. Here, we describe a Rosetta-based approach for both cryoEM and X-ray crystallography refinement of glycoproteins that is capable of correcting conformational and configurational errors in carbohydrates. Building upon a previous Rosetta framework, we introduced additional features and score terms enabling automatic detection, setup, and refinement of glycan-containing structures. We benchmarked this approach using 12 crystal structures and showed that glycan geometries can be automatically improved while maintaining good fit to the crystallographic data. Finally, we used this method to refine carbohydrates of the human coronavirus NL63 spike glycoprotein and of an HIV envelope glycoprotein, demonstrating its usefulness for cryoEM refinement.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30568167,

title = {Structure of the type VI secretion system TssK-TssF-TssG baseplate subcomplex revealed by cryo-electron microscopy},

author = {Young-Jun Park and Kaitlyn D Lacourse and Christian Cambillau and Frank DiMaio and Joseph D Mougous and David Veesler},

doi = {10.1038/s41467-018-07796-5},

issn = {2041-1723},

year  = {2018},

date = {2018-12-01},

journal = {Nat Commun},

volume = {9},

number = {1},

pages = {5385},

abstract = {Type VI secretion systems (T6SSs) translocate effectors into target cells and are made of a contractile sheath and a tube docked onto a multi-protein transmembrane complex via a baseplate. Although some information is available about the mechanisms of tail contraction leading to effector delivery, the detailed architecture and function of the baseplate remain unknown. Here, we report the 3.7 Å resolution cryo-electron microscopy reconstruction of an enteroaggregative Escherichia coli baseplate subcomplex assembled from TssK, TssF and TssG. The structure reveals two TssK trimers interact with a locally pseudo-3-fold symmetrical complex comprising two copies of TssF and one copy of TssG. TssF and TssG are structurally related to each other and to components of the phage T4 baseplate and of the type IV secretion system, strengthening the evolutionary relationships among these macromolecular machines. These results, together with bacterial two-hybrid assays, provide a structural framework to understand the T6SS baseplate architecture.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30327422,

title = {Cryo-electron microscopy structure of the lipid droplet-formation protein seipin},

author = {Xuewu Sui and Henning Arlt and Kelly P Brock and Zon Weng Lai and Frank DiMaio and Debora S Marks and Maofu Liao and Robert V Farese and Tobias C Walther},

doi = {10.1083/jcb.201809067},

issn = {1540-8140},

year  = {2018},

date = {2018-12-01},

journal = {J Cell Biol},

volume = {217},

number = {12},

pages = {4080--4091},

abstract = {Metabolic energy is stored in cells primarily as triacylglycerols in lipid droplets (LDs), and LD dysregulation leads to metabolic diseases. The formation of monolayer-bound LDs from the endoplasmic reticulum (ER) bilayer is poorly understood, but the ER protein seipin is essential to this process. In this study, we report a cryo-electron microscopy structure and functional characterization of  seipin. The structure reveals a ring-shaped dodecamer with the luminal domain of each monomer resolved at ∼4.0 Å. Each luminal domain monomer exhibits two distinctive features: a hydrophobic helix (HH) positioned toward the ER bilayer and a β-sandwich domain with structural similarity to lipid-binding proteins. This structure and our functional testing in cells suggest a model in which seipin oligomers initially detect forming LDs in the ER via HHs and subsequently act as membrane anchors to enable lipid transfer and LD growth.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30403372,

title = {Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core},

author = {Andrew J Borst and Connor E Weidle and Matthew D Gray and Brandon Frenz and Joost Snijder and M Gordon Joyce and Ivelin S Georgiev and Guillaume Be Stewart-Jones and Peter D Kwong and Andrew T McGuire and Frank DiMaio and Leonidas Stamatatos and Marie Pancera and David Veesler},

doi = {10.7554/eLife.37688},

issn = {2050-084X},

year  = {2018},

date = {2018-11-01},

journal = {Elife},

volume = {7},

abstract = {VRC01 broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env). Unlike mature antibodies, corresponding VRC01 germline precursors poorly bind to Env. Immunogen design has mostly relied on glycan removal from trimeric Env constructs and has had limited success in eliciting mature VRC01 bnAbs. To better understand elicitation of such bnAbs, we characterized the inferred germline precursor of VRC01 in complex with a modified trimeric 426c Env by cryo-electron microscopy and a 426c gp120 core by X-ray crystallography, biolayer interferometry, immunoprecipitation, and glycoproteomics. Our results show VRC01 germline antibodies interacted with a wild-type 426c core lacking variable loops 1-3 in the presence and absence of a glycan at position Asn276, with the latter form binding with higher affinity than the former. Interactions in the presence of an Asn276 oligosaccharide could be enhanced upon carbohydrate shortening, which should be considered for immunogen design.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30318141,

title = {Structural Insights into Mdn1, an Essential AAA Protein Required for Ribosome Biogenesis},

author = {Zhen Chen and Hiroshi Suzuki and Yuki Kobayashi and Ashley C Wang and Frank DiMaio and Shigehiro A Kawashima and Thomas Walz and Tarun M Kapoor},

doi = {10.1016/j.cell.2018.09.015},

issn = {1097-4172},

year  = {2018},

date = {2018-10-01},

journal = {Cell},

volume = {175},

number = {3},

pages = {822--834.e18},

abstract = {Mdn1 is an essential AAA (ATPase associated with various activities) protein that removes assembly factors from distinct precursors of the ribosomal 60S subunit. However, Mdn1's large size (∼5,000 amino acid [aa]) and its limited homology to other well-studied proteins have restricted our understanding of its remodeling function. Here, we present structures for S. pombe Mdn1 in the presence of AMPPNP at up to ∼4 Å or ATP plus Rbin-1, a chemical inhibitor, at ∼8 Å resolution. These data reveal that Mdn1's MIDAS domain is tethered to its ring-shaped AAA domain through an ∼20 nm long structured linker and a flexible ∼500 aa Asp/Glu-rich motif. We find that the MIDAS domain, which also binds other ribosome-assembly factors, docks onto the AAA ring in a nucleotide state-specific manner. Together, our findings reveal how conformational changes in the AAA ring can be directly transmitted to the MIDAS domain and thereby drive the targeted release of assembly factors from ribosomal 60S-subunit precursors.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29864529,

title = {cryoem-cloud-tools: A software platform to deploy and manage cryo-EM jobs in the cloud},

author = {Michael A Cianfrocco and Indrajit Lahiri and Frank DiMaio and Andres E Leschziner},

doi = {10.1016/j.jsb.2018.05.014},

issn = {1095-8657},

year  = {2018},

date = {2018-09-01},

journal = {J Struct Biol},

volume = {203},

number = {3},

pages = {230--235},

abstract = {Access to streamlined computational resources remains a significant bottleneck for new users of cryo-electron microscopy (cryo-EM). To address this, we have developed tools that will submit cryo-EM analysis routines and atomic model building jobs directly to Amazon Web Services (AWS) from a local computer or laptop. These new software tools ("cryoem-cloud-tools") have incorporated optimal data movement, security, and cost-saving strategies, giving novice users access to complex cryo-EM data processing pipelines. Integrating these tools into the RELION processing pipeline and graphical user interface we determined a 2.2 Å structure of ß-galactosidase in ∼55 h on AWS. We implemented a similar strategy to submit Rosetta atomic model building and refinement to AWS. These software tools dramatically reduce the barrier for entry of new users to cloud computing for cryo-EM and are freely available at cryoem-tools.cloud.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29748322,

title = {Near-atomic model of microtubule-tau interactions},

author = {Elizabeth H Kellogg and Nisreen M A Hejab and Simon Poepsel and Kenneth H Downing and Frank DiMaio and Eva Nogales},

doi = {10.1126/science.aat1780},

issn = {1095-9203},

year  = {2018},

date = {2018-06-01},

journal = {Science},

volume = {360},

number = {6394},

pages = {1242--1246},

abstract = {Tau is a developmentally regulated axonal protein that stabilizes and bundles microtubules (MTs). Its hyperphosphorylation is thought to cause detachment from MTs and subsequent aggregation into fibrils implicated in Alzheimer's disease. It is unclear which tau residues are crucial for tau-MT interactions, where tau binds on MTs, and how it stabilizes them. We used cryo-electron microscopy to visualize different tau constructs on MTs and computational approaches to generate atomic models of tau-tubulin interactions. The conserved tubulin-binding repeats within tau adopt similar extended structures along the crest of the protofilament, stabilizing the interface between tubulin dimers. Our structures explain the effect of phosphorylation on MT affinity and lead to a model of tau repeats binding in tandem along protofilaments, tethering together tubulin dimers and stabilizing polymerization interfaces.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29507254,

title = {Protein homology model refinement by large-scale energy optimization},

author = {Hahnbeom Park and Sergey Ovchinnikov and David E Kim and Frank DiMaio and David Baker},

doi = {10.1073/pnas.1719115115},

issn = {1091-6490},

year  = {2018},

date = {2018-03-01},

journal = {Proc Natl Acad Sci U S A},

volume = {115},

number = {12},

pages = {3054--3059},

abstract = {Proteins fold to their lowest free-energy structures, and hence the most straightforward way to increase the accuracy of a partially incorrect protein structure model is to search for the lowest-energy nearby structure. This direct approach has met with little success for two reasons: first, energy function inaccuracies can lead to false energy minima, resulting in model degradation rather than improvement; and second, even with an accurate energy function, the search problem is formidable because the energy only drops considerably in the immediate vicinity of the global minimum, and there are a very large number of degrees of freedom. Here we describe a large-scale energy optimization-based refinement method that incorporates advances in both search and energy function accuracy that can substantially improve the accuracy of low-resolution homology models. The method refined low-resolution homology models into correct folds for 50 of 84 diverse protein families and generated improved models in recent blind structure prediction experiments. Analyses of the basis for these improvements reveal contributions from both the improvements in conformational sampling techniques and the energy function.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29496880,

title = {Accurate computational design of multipass transmembrane proteins},

author = {Peilong Lu and Duyoung Min and Frank DiMaio and Kathy Y Wei and Michael D Vahey and Scott E Boyken and Zibo Chen and Jorge A Fallas and George Ueda and William Sheffler and Vikram Khipple Mulligan and Wenqing Xu and James U Bowie and David Baker},

doi = {10.1126/science.aaq1739},

issn = {1095-9203},

year  = {2018},

date = {2018-03-01},

journal = {Science},

volume = {359},

number = {6379},

pages = {1042--1046},

abstract = {The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable. Crystal structures of the designed dimer and tetramer-a rocket-shaped structure with a wide cytoplasmic base that funnels into eight transmembrane helices-are very close to the design models. Our results pave the way for the design of multispan membrane proteins with new functions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29440491,

title = {Cryo-EM structure of the bacterial actin AlfA reveals unique assembly and ATP-binding interactions and the absence of a conserved subdomain},

author = {Gülsima D Usluer and Frank DiMaio and Shun Kai Yang and Jesse M Hansen and Jessica K Polka and R Dyche Mullins and Justin M Kollman},

doi = {10.1073/pnas.1715836115},

issn = {1091-6490},

year  = {2018},

date = {2018-03-01},

journal = {Proc Natl Acad Sci U S A},

volume = {115},

number = {13},

pages = {3356--3361},

abstract = {Bacterial actins are an evolutionarily diverse family of ATP-dependent filaments built from protomers with a conserved structural fold. Actin-based segregation systems are encoded on many bacterial plasmids and function to partition plasmids into daughter cells. The bacterial actin AlfA segregates plasmids by a mechanism distinct from other partition systems, dependent on its unique dynamic properties. Here, we report the near-atomic resolution electron cryo-microscopy structure of the AlfA filament, which reveals a strikingly divergent filament architecture resulting from the loss of a subdomain conserved in all other actins and a mode of ATP binding. Its unusual assembly interfaces and nucleotide interactions provide insight into AlfA dynamics, and expand the range of evolutionary variation accessible to actin quaternary structure.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28940798,

title = {Protein structure prediction using Rosetta in CASP12},

author = {Sergey Ovchinnikov and Hahnbeom Park and David E Kim and Frank DiMaio and David Baker},

doi = {10.1002/prot.25390},

issn = {1097-0134},

year  = {2018},

date = {2018-03-01},

journal = {Proteins},

volume = {86 Suppl 1},

number = {Suppl 1},

pages = {113--121},

abstract = {We describe several notable aspects of our structure predictions using Rosetta in CASP12 in the free modeling (FM) and refinement (TR) categories. First, we had previously generated (and published) models for most large protein families lacking experimentally determined structures using Rosetta guided by co-evolution based contact predictions, and for several targets these models proved better starting points for comparative modeling than any known crystal structure-our model database thus starts to fulfill one of the goals of the original protein structure initiative. Second, while our "human" group simply submitted ROBETTA models for most targets, for six targets expert intervention improved predictions considerably; the largest improvement was for T0886 where we correctly parsed two discontinuous domains guided by predicted contact maps to accurately identify a structural homolog of the same fold. Third, Rosetta all atom refinement followed by MD simulations led to consistent but small improvements when starting models were close to the native structure, and larger but less consistent improvements when starting models were further away.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28913931,

title = {Automatic structure prediction of oligomeric assemblies using Robetta in CASP12},

author = {Hahnbeom Park and David E Kim and Sergey Ovchinnikov and David Baker and Frank DiMaio},

doi = {10.1002/prot.25387},

issn = {1097-0134},

year  = {2018},

date = {2018-03-01},

journal = {Proteins},

volume = {86 Suppl 1},

number = {Suppl 1},

pages = {283--291},

abstract = {Many naturally occurring protein systems function primarily as symmetric assemblies. Prediction of the quaternary structure of these assemblies is an important biological problem. This article describes automated tools we have developed for predicting the structures of symmetric protein assemblies in the Robetta structure prediction server. We assess the performance of this pipeline on a set of targets from the recent CASP12/CAPRI blind quaternary structure prediction experiment. Our approach successfully predicted 5 of 7 symmetric assemblies in this challenge, and was assessed as the best participating server group, and 1 of only 2 groups (human or server) with 2 predictions judged as high quality by the assessors. We also assess the method on a broader set of 22 natively symmetric CASP12 targets, where we show that oligomeric modeling can improve the accuracy of monomeric structure determination, particularly in highly intertwined oligomers.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29168508,

title = {Structural basis for the initiation of eukaryotic transcription-coupled DNA repair},

author = {Jun Xu and Indrajit Lahiri and Wei Wang and Adam Wier and Michael A Cianfrocco and Jenny Chong and Alissa A Hare and Peter B Dervan and Frank DiMaio and Andres E Leschziner and Dong Wang},

doi = {10.1038/nature24658},

issn = {1476-4687},

year  = {2017},

date = {2017-11-01},

journal = {Nature},

volume = {551},

number = {7682},

pages = {653--657},

abstract = {Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II-CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II-Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29033288,

title = {The Therapeutic Antibody LM609 Selectively Inhibits Ligand Binding to Human αβ Integrin via Steric Hindrance},

author = {Andrew J Borst and Zachary M James and William N Zagotta and Mark Ginsberg and Felix A Rey and Frank DiMaio and Marija Backovic and David Veesler},

doi = {10.1016/j.str.2017.09.007},

issn = {1878-4186},

year  = {2017},

date = {2017-11-01},

journal = {Structure},

volume = {25},

number = {11},

pages = {1732--1739.e5},

abstract = {The LM609 antibody specifically recognizes αβ integrin and inhibits angiogenesis, bone resorption, and viral infections in an arginine-glycine-aspartate-independent manner. LM609 entered phase II clinical trials for the treatment of several cancers and was also used for αβ-targeted radioimmunotherapy. To elucidate the mechanisms of recognition and inhibition of αβ integrin, we solved the structure of the LM609 antigen-binding fragment by X-ray crystallography and determined its binding affinity for αβ. Using single-particle electron microscopy, we show that LM609 binds at the interface between the β-propeller domain of the α chain and the βI domain of the β chain, near the RGD-binding site, of all observed integrin conformational states. Integrating these data with fluorescence size-exclusion chromatography, we demonstrate that LM609 sterically hinders access of large ligands to the RGD-binding pocket, without obstructing it. This work provides a structural framework to expedite future efforts utilizing LM609 as a diagnostic or therapeutic tool.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29035201,

title = {Anti-diabetic drug binding site in a mammalian K channel revealed by Cryo-EM},

author = {Gregory M Martin and Balamurugan Kandasamy and Frank DiMaio and Craig Yoshioka and Show-Ling Shyng},

doi = {10.7554/eLife.31054},

issn = {2050-084X},

year  = {2017},

date = {2017-10-01},

journal = {Elife},

volume = {6},

abstract = {Sulfonylureas are anti-diabetic medications that act by inhibiting pancreatic K channels composed of SUR1 and Kir6.2. The mechanism by which these drugs interact with and inhibit the channel has been extensively investigated, yet it remains unclear where the drug binding pocket resides. Here, we present a cryo-EM structure of a hamster SUR1/rat Kir6.2 channel bound to a high-affinity sulfonylurea drug glibenclamide and ATP at 3.63 Å resolution, which reveals unprecedented details of the ATP and glibenclamide binding sites. Importantly, the structure shows for the first time that glibenclamide is lodged in the transmembrane bundle of the SUR1-ABC core connected to the first nucleotide binding domain near the inner leaflet of the lipid bilayer. Mutation of residues predicted to interact with glibenclamide in our model led to reduced sensitivity to glibenclamide. Our structure provides novel mechanistic insights of how sulfonylureas and ATP interact with the K channel complex to inhibit channel activity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28759049,

title = {Structural basis of TIR-domain-assembly formation in MAL- and MyD88-dependent TLR4 signaling},

author = {Thomas Ve and Parimala R Vajjhala and Andrew Hedger and Tristan Croll and Frank DiMaio and Shane Horsefield and Xiong Yu and Peter Lavrencic and Zahid Hassan and Garry P Morgan and Ashley Mansell and Mehdi Mobli and Ailis O'Carroll and Brieuc Chauvin and Yann Gambin and Emma Sierecki and Michael J Landsberg and Katryn J Stacey and Edward H Egelman and Bostjan Kobe},

doi = {10.1038/nsmb.3444},

issn = {1545-9985},

year  = {2017},

date = {2017-09-01},

journal = {Nat Struct Mol Biol},

volume = {24},

number = {9},

pages = {743--751},

abstract = {Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28682307,

title = {Cryo-EM structure of the protein-conducting ERAD channel Hrd1 in complex with Hrd3},

author = {Stefan Schoebel and Wei Mi and Alexander Stein and Sergey Ovchinnikov and Ryan Pavlovicz and Frank DiMaio and David Baker and Melissa G Chambers and Huayou Su and Dongsheng Li and Tom A Rapoport and Maofu Liao},

doi = {10.1038/nature23314},

issn = {1476-4687},

year  = {2017},

date = {2017-08-01},

journal = {Nature},

volume = {548},

number = {7667},

pages = {352--355},

abstract = {Misfolded endoplasmic reticulum proteins are retro-translocated through the membrane into the cytosol, where they are poly-ubiquitinated, extracted from the membrane, and degraded by the proteasome-a pathway termed endoplasmic reticulum-associated protein degradation (ERAD). Proteins with misfolded domains in the endoplasmic reticulum lumen or membrane are discarded through the ERAD-L and ERAD-M pathways, respectively. In Saccharomyces cerevisiae, both pathways require the ubiquitin ligase Hrd1, a multi-spanning membrane protein with a cytosolic RING finger domain. Hrd1 is the crucial membrane component for retro-translocation, but it is unclear whether it forms a protein-conducting channel. Here we present a cryo-electron microscopy structure of S. cerevisiae Hrd1 in complex with its endoplasmic reticulum luminal binding partner, Hrd3. Hrd1 forms a dimer within the membrane with one or two Hrd3 molecules associated at its luminal side. Each Hrd1 molecule has eight transmembrane segments, five of which form an aqueous cavity extending from the cytosol almost to the endoplasmic reticulum lumen, while a segment of the neighbouring Hrd1 molecule forms a lateral seal. The aqueous cavity and lateral gate are reminiscent of features of protein-conducting conduits that facilitate polypeptide movement in the opposite direction-from the cytosol into or across membranes. Our results suggest that Hrd1 forms a retro-translocation channel for the movement of misfolded polypeptides through the endoplasmic reticulum membrane.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28628127,

title = {RosettaES: a sampling strategy enabling automated interpretation of difficult cryo-EM maps},

author = {Brandon Frenz and Alexandra C Walls and Edward H Egelman and David Veesler and Frank DiMaio},

doi = {10.1038/nmeth.4340},

issn = {1548-7105},

year  = {2017},

date = {2017-08-01},

journal = {Nat Methods},

volume = {14},

number = {8},

pages = {797--800},

abstract = {Accurate atomic modeling of macromolecular structures into cryo-electron microscopy (cryo-EM) maps is a major challenge, as the moderate resolution makes accurate placement of atoms difficult. We present Rosetta enumerative sampling (RosettaES), an automated tool that uses a fragment-based sampling strategy for de novo model completion of macromolecular structures from cryo-EM density maps at 3-5-Å resolution. On a benchmark set of nine proteins, RosettaES was able to identify near-native conformations in 85% of segments. RosettaES was also used to determine models for three challenging macromolecular structures.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28639939,

title = {Model for a novel membrane envelope in a filamentous hyperthermophilic virus},

author = {Peter Kasson and Frank DiMaio and Xiong Yu and Soizick Lucas-Staat and Mart Krupovic and Stefan Schouten and David Prangishvili and Edward H Egelman},

doi = {10.7554/eLife.26268},

issn = {2050-084X},

year  = {2017},

date = {2017-06-01},

journal = {Elife},

volume = {6},

abstract = {Biological membranes create compartments, and are usually formed by lipid bilayers. However, in hyperthermophilic archaea that live optimally at temperatures above 80°C the membranes are monolayers which resemble fused bilayers. Many double-stranded DNA viruses which parasitize such hosts, including the filamentous virus AFV1 of , are enveloped with a lipid-containing membrane. Using cryo-EM, we show that the membrane in AFV1 is a ~2 nm-thick monolayer, approximately half the expected membrane thickness, formed by host membrane-derived lipids which adopt a U-shaped 'horseshoe' conformation. We hypothesize that this unusual viral envelope structure results from the extreme curvature of the viral capsid, as 'horseshoe' lipid conformations favor such curvature and host membrane lipids that permit horseshoe conformations are selectively recruited into the viral envelope. The unusual envelope found in AFV1 also has many implications for biotechnology, since this membrane can survive the most aggressive conditions involving extremes of temperature and pH.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28430426,

title = {The Rosetta All-Atom Energy Function for Macromolecular Modeling and Design},

author = {Rebecca F Alford and Andrew Leaver-Fay and Jeliazko R Jeliazkov and Matthew J O'Meara and Frank P DiMaio and Hahnbeom Park and Maxim V Shapovalov and P Douglas Renfrew and Vikram K Mulligan and Kalli Kappel and Jason W Labonte and Michael S Pacella and Richard Bonneau and Philip Bradley and Roland L Dunbrack and Rhiju Das and David Baker and Brian Kuhlman and Tanja Kortemme and Jeffrey J Gray},

doi = {10.1021/acs.jctc.7b00125},

issn = {1549-9626},

year  = {2017},

date = {2017-06-01},

journal = {J Chem Theory Comput},

volume = {13},

number = {6},

pages = {3031--3048},

abstract = {Over the past decade, the Rosetta biomolecular modeling suite has informed diverse biological questions and engineering challenges ranging from interpretation of low-resolution structural data to design of nanomaterials, protein therapeutics, and vaccines. Central to Rosetta's success is the energy function: a model parametrized from small-molecule and X-ray crystal structure data used to approximate the energy associated with each biomolecule conformation. This paper describes the mathematical models and physical concepts that underlie the latest Rosetta energy function, called the Rosetta Energy Function 2015 (REF15). Applying these concepts, we explain how to use Rosetta energies to identify and analyze the features of biomolecular models. Finally, we discuss the latest advances in the energy function that extend its capabilities from soluble proteins to also include membrane proteins, peptides containing noncanonical amino acids, small molecules, carbohydrates, nucleic acids, and other macromolecules.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28396445,

title = {CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel},

author = {Zachary M James and Andrew J Borst and Yoni Haitin and Brandon Frenz and Frank DiMaio and William N Zagotta and David Veesler},

doi = {10.1073/pnas.1700248114},

issn = {1091-6490},

year  = {2017},

date = {2017-04-01},

journal = {Proc Natl Acad Sci U S A},

volume = {114},

number = {17},

pages = {4430--4435},

abstract = {Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxyl-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from -which shares sequence similarity to eukaryotic CNG and HCN channels-in the presence of a saturating concentration of cAMP. A short S4-S5 linker connects nearby voltage-sensing and pore domains to produce a non-domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27667482,

title = {Modeling oblong proteins and water-mediated interfaces with RosettaDock in CAPRI rounds 28-35},

author = {Nicholas A Marze and Jeliazko R Jeliazkov and Shourya S Roy Burman and Scott E Boyken and Frank DiMaio and Jeffrey J Gray},

doi = {10.1002/prot.25168},

issn = {1097-0134},

year  = {2017},

date = {2017-03-01},

journal = {Proteins},

volume = {85},

number = {3},

pages = {479--486},

abstract = {The 28th-35th rounds of the Critical Assessment of PRotein Interactions (CAPRI) served as a practical benchmark for our RosettaDock protein-protein docking protocols, highlighting strengths and weaknesses of the approach. We achieved acceptable or better quality models in three out of 11 targets. For the two α-repeat protein-green fluorescent protein (αrep-GFP) complexes, we used a novel ellipsoidal partial-global docking method (Ellipsoidal Dock) to generate models with 2.2 Å/1.5 Å interface RMSD, capturing 49%/42% of the native contacts, for the 7-/5-repeat αrep complexes. For the DNase-immunity protein complex, we used a new predictor of hydrogen-bonding networks, HBNet with Bridging Waters, to place individual water models at the complex interface; models were generated with 1.8 Å interface RMSD and 12% native water contacts recovered. The targets for which RosettaDock failed to create an acceptable model were typically difficult in general, as six had no acceptable models submitted by any CAPRI predictor. The UCH-L5-RPN13 and UCH-L5-INO80G de-ubiquitinating enzyme-inhibitor complexes comprised inhibitors undergoing significant structural changes upon binding, with the partners being highly interwoven in the docked complexes. Our failure to predict the nucleosome-enzyme complex in Target 95 was largely due to tight constraints we placed on our model based on sparse biochemical data suggesting two specific cross-interface interactions, preventing the correct structure from being sampled. While RosettaDock's three successes show that it is a state-of-the-art docking method, the difficulties with highly flexible and multi-domain complexes highlight the need for better flexible docking and domain-assembly methods. Proteins 2017; 85:479-486. © 2016 Wiley Periodicals, Inc.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28573585,

title = {Rosetta Structure Prediction as a Tool for Solving Difficult Molecular Replacement Problems},

author = {Frank DiMaio},

doi = {10.1007/978-1-4939-7000-1_19},

issn = {1940-6029},

year  = {2017},

date = {2017-01-01},

journal = {Methods Mol Biol},

volume = {1607},

pages = {455--466},

abstract = {Molecular replacement (MR), a method for solving the crystallographic phase problem using phases derived from a model of the target structure, has proven extremely valuable, accounting for the vast majority of structures solved by X-ray crystallography. However, when the resolution of data is low, or the starting model is very dissimilar to the target protein, solving structures via molecular replacement may be very challenging. In recent years, protein structure prediction methodology has emerged as a powerful tool in model building and model refinement for difficult molecular replacement problems. This chapter describes some of the tools available in Rosetta for model building and model refinement specifically geared toward difficult molecular replacement cases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27667334,

title = {Crucial steps in the structure determination of a coronavirus spike glycoprotein using cryo-electron microscopy},

author = {Alexandra Walls and M Alejandra Tortorici and Berend-Jan Bosch and Brandon Frenz and Peter J M Rottier and Frank DiMaio and Felix A Rey and David Veesler},

doi = {10.1002/pro.3048},

issn = {1469-896X},

year  = {2017},

date = {2017-01-01},

journal = {Protein Sci},

volume = {26},

number = {1},

pages = {113--121},

abstract = {The tremendous pandemic potential of coronaviruses was demonstrated twice in the last 15 years by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion functions. Despite their biomedical importance, coronavirus S glycoproteins have proven difficult targets for structural characterization, precluding high-resolution studies of the biologically relevant trimer. Recent technological developments in single particle cryo-electron microscopy allowed us to determine the first structure of a coronavirus S glycoprotein trimer which provided a framework to understand the mechanisms of viral entry and suggested potential inhibition strategies for this family of viruses. Here, we describe the key factors that enabled this breakthrough.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27766851,

title = {Simultaneous Optimization of Biomolecular Energy Functions on Features from Small Molecules and Macromolecules},

author = {Hahnbeom Park and Philip Bradley and Per Greisen and Yuan Liu and Vikram Khipple Mulligan and David E Kim and David Baker and Frank DiMaio},

doi = {10.1021/acs.jctc.6b00819},

issn = {1549-9626},

year  = {2016},

date = {2016-12-01},

journal = {J Chem Theory Comput},

volume = {12},

number = {12},

pages = {6201--6212},

abstract = {Most biomolecular modeling energy functions for structure prediction, sequence design, and molecular docking have been parametrized using existing macromolecular structural data; this contrasts molecular mechanics force fields which are largely optimized using small-molecule data. In this study, we describe an integrated method that enables optimization of a biomolecular modeling energy function simultaneously against small-molecule thermodynamic data and high-resolution macromolecular structural data. We use this approach to develop a next-generation Rosetta energy function that utilizes a new anisotropic implicit solvation model, and an improved electrostatics and Lennard-Jones model, illustrating how energy functions can be considerably improved in their ability to describe large-scale energy landscapes by incorporating both small-molecule and macromolecule data. The energy function improves performance in a wide range of protein structure prediction challenges, including monomeric structure prediction, protein-protein and protein-ligand docking, protein sequence design, and prediction of the free energy changes by mutation, while reasonably recapitulating small-molecule thermodynamic properties.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27746017,

title = {Cryo-EM Structure of Caspase-8 Tandem DED Filament Reveals Assembly and Regulation Mechanisms of the Death-Inducing Signaling Complex},

author = {Tian-Min Fu and Yang Li and Alvin Lu and Zongli Li and Parimala R Vajjhala and Anthony C Cruz and Devendra B Srivastava and Frank DiMaio and Pawel A Penczek and Richard M Siegel and Katryn J Stacey and Edward H Egelman and Hao Wu},

doi = {10.1016/j.molcel.2016.09.009},

issn = {1097-4164},

year  = {2016},

date = {2016-10-01},

journal = {Mol Cell},

volume = {64},

number = {2},

pages = {236--250},

abstract = {Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27617430,

title = {Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy},

author = {Alexandra C Walls and M Alejandra Tortorici and Brandon Frenz and Joost Snijder and Wentao Li and Félix A Rey and Frank DiMaio and Berend-Jan Bosch and David Veesler},

doi = {10.1038/nsmb.3293},

issn = {1545-9985},

year  = {2016},

date = {2016-10-01},

journal = {Nat Struct Mol Biol},

volume = {23},

number = {10},

pages = {899--905},

abstract = {The threat of a major coronavirus pandemic urges the development of strategies to combat these pathogens. Human coronavirus NL63 (HCoV-NL63) is an α-coronavirus that can cause severe lower-respiratory-tract infections requiring hospitalization. We report here the 3.4-Å-resolution cryo-EM reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which mediates entry into host cells and is the main target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass spectrometry, provides a structural framework to understand the accessibility to antibodies. The structure reveals the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on epitope masking with glycans and activating conformational changes, to evade the immune system of infected hosts.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27669148,

title = {Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta},

author = {Ray Yu-Ruei Wang and Yifan Song and Benjamin A Barad and Yifan Cheng and James S Fraser and Frank DiMaio},

doi = {10.7554/eLife.17219},

issn = {2050-084X},

year  = {2016},

date = {2016-09-01},

journal = {Elife},

volume = {5},

abstract = {Cryo-EM has revealed the structures of many challenging yet exciting macromolecular assemblies at near-atomic resolution (3-4.5Å), providing biological phenomena with molecular descriptions. However, at these resolutions, accurately positioning individual atoms remains challenging and error-prone. Manually refining thousands of amino acids - typical in a macromolecular assembly - is tedious and time-consuming. We present an automated method that can improve the atomic details in models that are manually built in near-atomic-resolution cryo-EM maps. Applying the method to three systems recently solved by cryo-EM, we are able to improve model geometry while maintaining the fit-to-density. Backbone placement errors are automatically detected and corrected, and the refinement shows a large radius of convergence. The results demonstrate that the method is amenable to structures with symmetry, of very large size, and containing RNA as well as covalently bound ligands. The method should streamline the cryo-EM structure determination process, providing accurate and unbiased atomic structure interpretation of such maps.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26677056,

title = {Improved de novo structure prediction in CASP11 by incorporating coevolution information into Rosetta},

author = {Sergey Ovchinnikov and David E Kim and Ray Yu-Ruei Wang and Yuan Liu and Frank DiMaio and David Baker},

doi = {10.1002/prot.24974},

issn = {1097-0134},

year  = {2016},

date = {2016-09-01},

journal = {Proteins},

volume = {84 Suppl 1},

number = {Suppl 1},

pages = {67--75},

abstract = {We describe CASP11 de novo blind structure predictions made using the Rosetta structure prediction methodology with both automatic and human assisted protocols. Model accuracy was generally improved using coevolution derived residue-residue contact information as restraints during Rosetta conformational sampling and refinement, particularly when the number of sequences in the family was more than three times the length of the protein. The highlight was the human assisted prediction of T0806, a large and topologically complex target with no homologs of known structure, which had unprecedented accuracy-<3.0 Å root-mean-square deviation (RMSD) from the crystal structure over 223 residues. For this target, we increased the amount of conformational sampling over our fully automated method by employing an iterative hybridization protocol. Our results clearly demonstrate, in a blind prediction scenario, that coevolution derived contacts can considerably increase the accuracy of template-free structure modeling. Proteins 2016; 84(Suppl 1):67-75. © 2015 Wiley Periodicals, Inc.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26205421,

title = {CASP11 refinement experiments with ROSETTA},

author = {Hahnbeom Park and Frank DiMaio and David Baker},

doi = {10.1002/prot.24862},

issn = {1097-0134},

year  = {2016},

date = {2016-09-01},

journal = {Proteins},

volume = {84 Suppl 1},

number = {Suppl 1},

pages = {314--322},

abstract = {We report new Rosetta-based approaches to tackling the major issues that confound protein structure refinement, and the testing of these approaches in the CASP11 experiment. Automated refinement protocols were developed that integrate a range of sampling methods using parallel computation and multiobjective optimization. In CASP11, we used a more aggressive large-scale structure rebuilding approach for poor starting models, and a less aggressive local rebuilding plus core refinement approach for starting models likely to be closer to the native structure. The more incorrectly modeled a structure was predicted to be, the more it was allowed to vary during refinement. The CASP11 experiment revealed strengths and weaknesses of the approaches: the high-resolution strategy incorporating local rebuilding with core refinement consistently improved starting structures, while the low-resolution strategy incorporating the reconstruction of large parts of the structures improved starting models in some cases but often considerably worsened them, largely because of model selection issues. Overall, the results suggest the high-resolution refinement protocol is a promising method orthogonal to other approaches, while the low-resolution refinement method clearly requires further development. Proteins 2016; 84(Suppl 1):314-322. © 2015 Wiley Periodicals, Inc.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26205421b,

title = {CASP11 refinement experiments with ROSETTA},

author = {Hahnbeom Park and Frank DiMaio and David Baker},

doi = {10.1002/prot.24862},

issn = {1097-0134},

year  = {2016},

date = {2016-09-01},

journal = {Proteins},

volume = {84 Suppl 1},

number = {Suppl 1},

pages = {314--322},

abstract = {We report new Rosetta-based approaches to tackling the major issues that confound protein structure refinement, and the testing of these approaches in the CASP11 experiment. Automated refinement protocols were developed that integrate a range of sampling methods using parallel computation and multiobjective optimization. In CASP11, we used a more aggressive large-scale structure rebuilding approach for poor starting models, and a less aggressive local rebuilding plus core refinement approach for starting models likely to be closer to the native structure. The more incorrectly modeled a structure was predicted to be, the more it was allowed to vary during refinement. The CASP11 experiment revealed strengths and weaknesses of the approaches: the high-resolution strategy incorporating local rebuilding with core refinement consistently improved starting structures, while the low-resolution strategy incorporating the reconstruction of large parts of the structures improved starting models in some cases but often considerably worsened them, largely because of model selection issues. Overall, the results suggest the high-resolution refinement protocol is a promising method orthogonal to other approaches, while the low-resolution refinement method clearly requires further development. Proteins 2016; 84(Suppl 1):314-322. © 2015 Wiley Periodicals, Inc.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27490953,

title = {Protocols for Molecular Modeling with Rosetta3 and RosettaScripts},

author = {Brian J Bender and Alberto Cisneros and Amanda M Duran and Jessica A Finn and Darwin Fu and Alyssa D Lokits and Benjamin K Mueller and Amandeep K Sangha and Marion F Sauer and Alexander M Sevy and Gregory Sliwoski and Jonathan H Sheehan and Frank DiMaio and Jens Meiler and Rocco Moretti},

doi = {10.1021/acs.biochem.6b00444},

issn = {1520-4995},

year  = {2016},

date = {2016-08-01},

journal = {Biochemistry},

volume = {55},

number = {34},

pages = {4748--4763},

abstract = {Previously, we published an article providing an overview of the Rosetta suite of biomacromolecular modeling software and a series of step-by-step tutorials [Kaufmann, K. W., et al. (2010) Biochemistry 49, 2987-2998]. The overwhelming positive response to this publication we received motivates us to here share the next iteration of these tutorials that feature de novo folding, comparative modeling, loop construction, protein docking, small molecule docking, and protein design. This updated and expanded set of tutorials is needed, as since 2010 Rosetta has been fully redesigned into an object-oriented protein modeling program Rosetta3. Notable improvements include a substantially improved energy function, an XML-like language termed "RosettaScripts" for flexibly specifying modeling task, new analysis tools, the addition of the TopologyBroker to control conformational sampling, and support for multiple templates in comparative modeling. Rosetta's ability to model systems with symmetric proteins, membrane proteins, noncanonical amino acids, and RNA has also been greatly expanded and improved.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27239808,

title = {Improving hybrid statistical and physical forcefields through local structure enumeration},

author = {Patrick Conway and Frank DiMaio},

doi = {10.1002/pro.2956},

issn = {1469-896X},

year  = {2016},

date = {2016-08-01},

journal = {Protein Sci},

volume = {25},

number = {8},

pages = {1525--1534},

abstract = {Forcefields used in biomolecular simulations are comprised of energetic terms that are physical in nature, based on parameter fitting to quantum mechanical simulation or experimental data, or statistical, drawing off high-resolution structural data to describe distributions of molecular features. Combining the two in a single forcefield is challenging, since physical terms describe some, but not all, of the observed statistics, leading to double counting. In this manuscript, we develop a general scheme for correcting statistical potentials used in combination with physical terms. We apply these corrections to the sidechain torsional potential used in the Rosetta all-atom forcefield. We show the approach identifies instances of double-counted interactions, including electrostatic interactions between sidechain and nearby backbone, and steric interactions between neighboring Cβ atoms within secondary structural elements. Moreover, this scheme allows for the inclusion of intraresidue physical terms, previously turned off to avoid overlap with the statistical potential. Combined, these corrections lead to a forcefield with improved performance on several structure prediction tasks, including rotamer prediction and native structure discrimination.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27151862,

title = {De novo design of protein homo-oligomers with modular hydrogen-bond network-mediated specificity},

author = {Scott E Boyken and Zibo Chen and Benjamin Groves and Robert A Langan and Gustav Oberdorfer and Alex Ford and Jason M Gilmore and Chunfu Xu and Frank DiMaio and Jose Henrique Pereira and Banumathi Sankaran and Georg Seelig and Peter H Zwart and David Baker},

doi = {10.1126/science.aad8865},

issn = {1095-9203},

year  = {2016},

date = {2016-05-01},

journal = {Science},

volume = {352},

number = {6286},

pages = {680--687},

abstract = {In nature, structural specificity in DNA and proteins is encoded differently: In DNA, specificity arises from modular hydrogen bonds in the core of the double helix, whereas in proteins, specificity arises largely from buried hydrophobic packing complemented by irregular peripheral polar interactions. Here, we describe a general approach for designing a wide range of protein homo-oligomers with specificity determined by modular arrays of central hydrogen-bond networks. We use the approach to design dimers, trimers, and tetramers consisting of two concentric rings of helices, including previously not seen triangular, square, and supercoiled topologies. X-ray crystallography confirms that the structures overall, and the hydrogen-bond networks in particular, are nearly identical to the design models, and the networks confer interaction specificity in vivo. The ability to design extensive hydrogen-bond networks with atomic accuracy enables the programming of protein interaction specificity for a broad range of synthetic biology applications; more generally, our results demonstrate that, even with the tremendous diversity observed in nature, there are fundamentally new modes of interaction to be discovered in proteins.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26855426,

title = {Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer},

author = {Alexandra C Walls and M Alejandra Tortorici and Berend-Jan Bosch and Brandon Frenz and Peter J M Rottier and Frank DiMaio and Félix A Rey and David Veesler},

doi = {10.1038/nature16988},

issn = {1476-4687},

year  = {2016},

date = {2016-03-01},

journal = {Nature},

volume = {531},

number = {7592},

pages = {114--117},

abstract = {The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion functions. S also contains the principal antigenic determinants and is the target of neutralizing antibodies. Here we present the structure of a mouse coronavirus S trimer ectodomain determined at 4.0 Å resolution by single particle cryo-electron microscopy. It reveals the metastable pre-fusion architecture of S and highlights key interactions stabilizing it. The structure shares a common core with paramyxovirus F proteins, implicating mechanistic similarities and an evolutionary connection between these viral fusion proteins. The accessibility of the highly conserved fusion peptide at the periphery of the trimer indicates potential vaccinology strategies to elicit broadly neutralizing antibodies against coronaviruses. Finally, comparison with crystal structures of human coronavirus S domains allows rationalization of the molecular basis for species specificity based on the use of spatially contiguous but distinct domains.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27572730,

title = {Tools for Model Building and Optimization into Near-Atomic Resolution Electron Cryo-Microscopy Density Maps},

author = {F DiMaio and W Chiu},

doi = {10.1016/bs.mie.2016.06.003},

issn = {1557-7988},

year  = {2016},

date = {2016-01-01},

journal = {Methods Enzymol},

volume = {579},

pages = {255--276},

abstract = {Electron cryo-microscopy (cryoEM) has advanced dramatically to become a viable tool for high-resolution structural biology research. The ultimate outcome of a cryoEM study is an atomic model of a macromolecule or its complex with interacting partners. This chapter describes a variety of algorithms and software to build a de novo model based on the cryoEM 3D density map, to optimize the model with the best stereochemistry restraints and finally to validate the model with proper protocols. The full process of atomic structure determination from a cryoEM map is described. The tools outlined in this chapter should prove extremely valuable in revealing atomic interactions guided by cryoEM data.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26724865,

title = {Structure of a Chaperone-Usher Pilus Reveals the Molecular Basis of Rod Uncoiling},

author = {Manuela K Hospenthal and Adam Redzej and Karen Dodson and Marta Ukleja and Brandon Frenz and Catarina Rodrigues and Scott J Hultgren and Frank DiMaio and Edward H Egelman and Gabriel Waksman},

doi = {10.1016/j.cell.2015.11.049},

issn = {1097-4172},

year  = {2016},

date = {2016-01-01},

journal = {Cell},

volume = {164},

number = {1-2},

pages = {269--278},

abstract = {Types 1 and P pili are prototypical bacterial cell-surface appendages playing essential roles in mediating adhesion of bacteria to the urinary tract. These pili, assembled by the chaperone-usher pathway, are polymers of pilus subunits assembling into two parts: a thin, short tip fibrillum at the top, mounted on a long pilus rod. The rod adopts a helical quaternary structure and is thought to play essential roles: its formation may drive pilus extrusion by preventing backsliding of the nascent growing pilus within the secretion pore; the rod also has striking spring-like properties, being able to uncoil and recoil depending on the intensity of shear forces generated by urine flow. Here, we present an atomic model of the P pilus generated from a 3.8 Å resolution cryo-electron microscopy reconstruction. This structure provides the molecular basis for the rod's remarkable mechanical properties and illuminates its role in pilus secretion.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26724865b,

title = {Structure of a Chaperone-Usher Pilus Reveals the Molecular Basis of Rod Uncoiling},

author = {Manuela K Hospenthal and Adam Redzej and Karen Dodson and Marta Ukleja and Brandon Frenz and Catarina Rodrigues and Scott J Hultgren and Frank DiMaio and Edward H Egelman and Gabriel Waksman},

doi = {10.1016/j.cell.2015.11.049},

issn = {1097-4172},

year  = {2016},

date = {2016-01-01},

journal = {Cell},

volume = {164},

number = {1-2},

pages = {269--278},

abstract = {Types 1 and P pili are prototypical bacterial cell-surface appendages playing essential roles in mediating adhesion of bacteria to the urinary tract. These pili, assembled by the chaperone-usher pathway, are polymers of pilus subunits assembling into two parts: a thin, short tip fibrillum at the top, mounted on a long pilus rod. The rod adopts a helical quaternary structure and is thought to play essential roles: its formation may drive pilus extrusion by preventing backsliding of the nascent growing pilus within the secretion pore; the rod also has striking spring-like properties, being able to uncoil and recoil depending on the intensity of shear forces generated by urine flow. Here, we present an atomic model of the P pilus generated from a 3.8 Å resolution cryo-electron microscopy reconstruction. This structure provides the molecular basis for the rod's remarkable mechanical properties and illuminates its role in pilus secretion.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26650357,

title = {Precise assembly of complex beta sheet topologies from de novo designed building blocks},

author = {Indigo Chris King and James Gleixner and Lindsey Doyle and Alexandre Kuzin and John F Hunt and Rong Xiao and Gaetano T Montelione and Barry L Stoddard and Frank DiMaio and David Baker},

doi = {10.7554/eLife.11012},

issn = {2050-084X},

year  = {2015},

date = {2015-12-01},

journal = {Elife},

volume = {4},

abstract = {Design of complex alpha-beta protein topologies poses a challenge because of the large number of alternative packing arrangements. A similar challenge presumably limited the emergence of large and complex protein topologies in evolution. Here, we demonstrate that protein topologies with six and seven-stranded beta sheets can be designed by insertion of one de novo designed beta sheet containing protein into another such that the two beta sheets are merged to form a single extended sheet, followed by amino acid sequence optimization at the newly formed strand-strand, strand-helix, and helix-helix interfaces. Crystal structures of two such designs closely match the computational design models. Searches for similar structures in the SCOP protein domain database yield only weak matches with different beta sheet connectivities. A similar beta sheet fusion mechanism may have contributed to the emergence of complex beta sheets during natural protein evolution.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26650357b,

title = {Precise assembly of complex beta sheet topologies from de novo designed building blocks},

author = {Indigo Chris King and James Gleixner and Lindsey Doyle and Alexandre Kuzin and John F Hunt and Rong Xiao and Gaetano T Montelione and Barry L Stoddard and Frank DiMaio and David Baker},

doi = {10.7554/eLife.11012},

issn = {2050-084X},

year  = {2015},

date = {2015-12-01},

journal = {Elife},

volume = {4},

abstract = {Design of complex alpha-beta protein topologies poses a challenge because of the large number of alternative packing arrangements. A similar challenge presumably limited the emergence of large and complex protein topologies in evolution. Here, we demonstrate that protein topologies with six and seven-stranded beta sheets can be designed by insertion of one de novo designed beta sheet containing protein into another such that the two beta sheets are merged to form a single extended sheet, followed by amino acid sequence optimization at the newly formed strand-strand, strand-helix, and helix-helix interfaces. Crystal structures of two such designs closely match the computational design models. Searches for similar structures in the SCOP protein domain database yield only weak matches with different beta sheet connectivities. A similar beta sheet fusion mechanism may have contributed to the emergence of complex beta sheets during natural protein evolution.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26280328,

title = {EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy},

author = {Benjamin A Barad and Nathaniel Echols and Ray Yu-Ruei Wang and Yifan Cheng and Frank DiMaio and Paul D Adams and James S Fraser},

doi = {10.1038/nmeth.3541},

issn = {1548-7105},

year  = {2015},

date = {2015-10-01},

journal = {Nat Methods},

volume = {12},

number = {10},

pages = {943--946},

abstract = {Advances in high-resolution cryo-electron microscopy (cryo-EM) require the development of validation metrics to independently assess map quality and model geometry. We report EMRinger, a tool that assesses the precise fitting of an atomic model into the map during refinement and shows how radiation damage alters scattering from negatively charged amino acids. EMRinger (https://github.com/fraser-lab/EMRinger) will be useful for monitoring progress in resolving and modeling high-resolution features in cryo-EM.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26280328b,

title = {EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy},

author = {Benjamin A Barad and Nathaniel Echols and Ray Yu-Ruei Wang and Yifan Cheng and Frank DiMaio and Paul D Adams and James S Fraser},

doi = {10.1038/nmeth.3541},

issn = {1548-7105},

year  = {2015},

date = {2015-10-01},

journal = {Nat Methods},

volume = {12},

number = {10},

pages = {943--946},

abstract = {Advances in high-resolution cryo-electron microscopy (cryo-EM) require the development of validation metrics to independently assess map quality and model geometry. We report EMRinger, a tool that assesses the precise fitting of an atomic model into the map during refinement and shows how radiation damage alters scattering from negatively charged amino acids. EMRinger (https://github.com/fraser-lab/EMRinger) will be useful for monitoring progress in resolving and modeling high-resolution features in cryo-EM.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26167882,

title = {The molecular basis for flexibility in the flexible filamentous plant viruses},

author = {Frank DiMaio and Chun-Chieh Chen and Xiong Yu and Brandon Frenz and Yau-Heiu Hsu and Na-Sheng Lin and Edward H Egelman},

doi = {10.1038/nsmb.3054},

issn = {1545-9985},

year  = {2015},

date = {2015-08-01},

journal = {Nat Struct Mol Biol},

volume = {22},

number = {8},

pages = {642--644},

abstract = {Flexible filamentous plant viruses cause more than half the viral crop damage in the world but are also potentially useful for biotechnology. Structural studies began more than 75 years ago but have failed, owing to the virion's extreme flexibility. We have used cryo-EM to generate an atomic model for bamboo mosaic virus, which reveals flexible N- and C-terminal extensions that allow deformation while still maintaining structural integrity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25986921,

title = {Designing Two-Dimensional Protein Arrays through Fusion of Multimers and Interface Mutations},

author = {James F Matthaei and Frank DiMaio and Jeffrey J Richards and Lilo D Pozzo and David Baker and François Baneyx},

doi = {10.1021/acs.nanolett.5b01499},

issn = {1530-6992},

year  = {2015},

date = {2015-08-01},

journal = {Nano Lett},

volume = {15},

number = {8},

pages = {5235--5239},

abstract = {We have combined fusion of oligomers with cyclic symmetry and alanine substitutions to eliminate clashes and produce proteins that self-assemble into 2-D arrays upon addition of calcium ions. Using TEM, AFM, small-angle X-ray scattering, and fluorescence microscopy, we show that the designed lattices which are 5 nm high with p3 space group symmetry and 7.25 nm periodicity self-assemble into structures that can exceed 100 μm in characteristic length. The versatile strategy, experimental approach, and hexagonal arrays described herein should prove valuable for the engineering of functional nanostructured materials in 2-D.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26170309,

title = {Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy},

author = {Neil B Blok and Dongyan Tan and Ray Yu-Ruei Wang and Pawel A Penczek and David Baker and Frank DiMaio and Tom A Rapoport and Thomas Walz},

doi = {10.1073/pnas.1500257112},

issn = {1091-6490},

year  = {2015},

date = {2015-07-01},

journal = {Proc Natl Acad Sci U S A},

volume = {112},

number = {30},

pages = {E4017--E4025},

abstract = {Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the Pex1/Pex6 complex; mutations in these proteins frequently cause peroxisomal diseases. The structures were determined in the presence of different nucleotides by cryo-electron microscopy. Models were generated using a computational approach that combines Monte Carlo placement of structurally homologous domains into density maps with energy minimization and refinement protocols. Pex1 and Pex6 alternate in an unprecedented hexameric double ring. Each protein has two N-terminal domains, N1 and N2, structurally related to the single N domains in p97 and N-ethylmaleimide sensitive factor (NSF); N1 of Pex1 is mobile, but the others are packed against the double ring. The N-terminal ATPase domains are inactive, forming a symmetric D1 ring, whereas the C-terminal domains are active, likely in different nucleotide states, and form an asymmetric D2 ring. These results suggest how subunit activity is coordinated and indicate striking similarities between Pex1/Pex6 and p97, supporting the hypothesis that the Pex1/Pex6 complex has a role in peroxisomal protein import analogous to p97 in ER-associated protein degradation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26089516,

title = {Design of ordered two-dimensional arrays mediated by noncovalent protein-protein interfaces},

author = {Shane Gonen and Frank DiMaio and Tamir Gonen and David Baker},

doi = {10.1126/science.aaa9897},

issn = {1095-9203},

year  = {2015},

date = {2015-06-01},

journal = {Science},

volume = {348},

number = {6241},

pages = {1365--1368},

abstract = {We describe a general approach to designing two-dimensional (2D) protein arrays mediated by noncovalent protein-protein interfaces. Protein homo-oligomers are placed into one of the seventeen 2D layer groups, the degrees of freedom of the lattice are sampled to identify configurations with shape-complementary interacting surfaces, and the interaction energy is minimized using sequence design calculations. We used the method to design proteins that self-assemble into layer groups P 3 2 1, P 4 2(1) 2, and P 6. Projection maps of micrometer-scale arrays, assembled both in vitro and in vivo, are consistent with the design models and display the target layer group symmetry. Such programmable 2D protein lattices should enable new approaches to structure determination, sensing, and nanomaterial engineering.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25960407,

title = {The origin of consistent protein structure refinement from structural averaging},

author = {Hahnbeom Park and Frank DiMaio and David Baker},

doi = {10.1016/j.str.2015.03.022},

issn = {1878-4186},

year  = {2015},

date = {2015-06-01},

journal = {Structure},

volume = {23},

number = {6},

pages = {1123--1128},

abstract = {Recent studies have shown that explicit solvent molecular dynamics (MD) simulation followed by structural averaging can consistently improve protein structure models. We find that improvement upon averaging is not limited to explicit water MD simulation, as consistent improvements are also observed for more efficient implicit solvent MD or Monte Carlo minimization simulations. To determine the origin of these improvements, we examine the changes in model accuracy brought about by averaging at the individual residue level. We find that the improvement in model quality from averaging results from the superposition of two effects: a dampening of deviations from the correct structure in the least well modeled regions, and a reinforcement of consistent movements towards the correct structure in better modeled regions. These observations are consistent with an energy landscape model in which the magnitude of the energy gradient toward the native structure decreases with increasing distance from the native state.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25999507,

title = {Virology. A virus that infects a hyperthermophile encapsidates A-form DNA},

author = {Frank DiMaio and Xiong Yu and Elena Rensen and Mart Krupovic and David Prangishvili and Edward H Egelman},

doi = {10.1126/science.aaa4181},

issn = {1095-9203},

year  = {2015},

date = {2015-05-01},

journal = {Science},

volume = {348},

number = {6237},

pages = {914--917},

abstract = {Extremophiles, microorganisms thriving in extreme environmental conditions, must have proteins and nucleic acids that are stable at extremes of temperature and pH. The nonenveloped, rod-shaped virus SIRV2 (Sulfolobus islandicus rod-shaped virus 2) infects the hyperthermophilic acidophile Sulfolobus islandicus, which lives at 80°C and pH 3. We have used cryo-electron microscopy to generate a three-dimensional reconstruction of the SIRV2 virion at ~4 angstrom resolution, which revealed a previously unknown form of virion organization. Although almost half of the capsid protein is unstructured in solution, this unstructured region folds in the virion into a single extended α helix that wraps around the DNA. The DNA is entirely in the A-form, which suggests a common mechanism with bacterial spores for protecting DNA in the most adverse environments.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25707030,

title = {Atomic-accuracy models from 4.5-Å cryo-electron microscopy data with density-guided iterative local refinement},

author = {Frank DiMaio and Yifan Song and Xueming Li and Matthias J Brunner and Chunfu Xu and Vincent Conticello and Edward Egelman and Thomas Marlovits and Yifan Cheng and David Baker},

doi = {10.1038/nmeth.3286},

issn = {1548-7105},

year  = {2015},

date = {2015-04-01},

journal = {Nat Methods},

volume = {12},

number = {4},

pages = {361--365},

abstract = {We describe a general approach for refining protein structure models on the basis of cryo-electron microscopy maps with near-atomic resolution. The method integrates Monte Carlo sampling with local density-guided optimization, Rosetta all-atom refinement and real-space B-factor fitting. In tests on experimental maps of three different systems with 4.5-Å resolution or better, the method consistently produced models with atomic-level accuracy largely independently of starting-model quality, and it outperformed the molecular dynamics-based MDFF method. Cross-validated model quality statistics correlated with model accuracy over the three test systems.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25707029,

title = {De novo protein structure determination from near-atomic-resolution cryo-EM maps},

author = {Ray Yu-Ruei Wang and Mikhail Kudryashev and Xueming Li and Edward H Egelman and Marek Basler and Yifan Cheng and David Baker and Frank DiMaio},

doi = {10.1038/nmeth.3287},

issn = {1548-7105},

year  = {2015},

date = {2015-04-01},

journal = {Nat Methods},

volume = {12},

number = {4},

pages = {335--338},

abstract = {We present a de novo model-building approach that combines predicted backbone conformations with side-chain fit to density to accurately assign sequence into density maps. This method yielded accurate models for six of nine experimental maps at 3.3- to 4.8-Å resolution and produced a nearly complete model for an unsolved map containing a 660-residue heterodimeric protein. This method should enable rapid and reliable protein structure determination from near-atomic-resolution cryo-electron microscopy (cryo-EM) maps.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25684576,

title = {Structure of EspB from the ESX-1 type VII secretion system and insights into its export mechanism},

author = {Matthew Solomonson and Dheva Setiaputra and Karl A T Makepeace and Emilie Lameignere and Evgeniy V Petrotchenko and Deborah G Conrady and Julien R Bergeron and Marija Vuckovic and Frank DiMaio and Christoph H Borchers and Calvin K Yip and Natalie C J Strynadka},

doi = {10.1016/j.str.2015.01.002},

issn = {1878-4186},

year  = {2015},

date = {2015-03-01},

journal = {Structure},

volume = {23},

number = {3},

pages = {571--583},

abstract = {Mycobacterium tuberculosis (Mtb) uses the ESX-1 type VII secretion system to export virulence proteins across its lipid-rich cell wall, which helps permeabilize the host's macrophage phagosomal membrane, facilitating the escape and cell-to-cell spread of Mtb. ESX-1 membranolytic activity depends on a set of specialized secreted Esp proteins, the structure and specific roles of which are not currently understood. Here, we report the X-ray and electron microscopic structures of the ESX-1-secreted EspB. We demonstrate that EspB adopts a PE/PPE-like fold that mediates oligomerization with apparent heptameric symmetry, generating a barrel-shaped structure with a central pore that we propose contributes to the macrophage killing functions of EspB. Our structural data also reveal unexpected direct interactions between the EspB bipartite secretion signal sequence elements that form a unified aromatic surface. These findings provide insight into how specialized proteins encoded within the ESX-1 locus are targeted for secretion, and for the first time indicate an oligomerization-dependent role for Esp virulence factors.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25866491,

title = {Combined covalent-electrostatic model of hydrogen bonding improves structure prediction with Rosetta},

author = {Matthew J O'Meara and Andrew Leaver-Fay and Michael D Tyka and Amelie Stein and Kevin Houlihan and Frank DiMaio and Philip Bradley and Tanja Kortemme and David Baker and Jack Snoeyink and Brian Kuhlman},

doi = {10.1021/ct500864r},

issn = {1549-9626},

year  = {2015},

date = {2015-02-01},

journal = {J Chem Theory Comput},

volume = {11},

number = {2},

pages = {609--622},

abstract = {Interactions between polar atoms are challenging to model because at very short ranges they form hydrogen bonds (H-bonds) that are partially covalent in character and exhibit strong orientation preferences; at longer ranges the orientation preferences are lost, but significant electrostatic interactions between charged and partially charged atoms remain. To simultaneously model these two types of behavior, we refined an orientation dependent model of hydrogen bonds [Kortemme et al. J. Mol. Biol. 2003, 326, 1239] used by the molecular modeling program Rosetta and then combined it with a distance-dependent Coulomb model of electrostatics. The functional form of the H-bond potential is physically motivated and parameters are fit so that H-bond geometries that Rosetta generates closely resemble H-bond geometries in high-resolution crystal structures. The combined potentials improve performance in a variety of scientific benchmarks including decoy discrimination, side chain prediction, and native sequence recovery in protein design simulations and establishes a new standard energy function for Rosetta.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25723169,

title = {Structure of the type VI secretion system contractile sheath},

author = {Mikhail Kudryashev and Ray Yu-Ruei Wang and Maximilian Brackmann and Sebastian Scherer and Timm Maier and David Baker and Frank DiMaio and Henning Stahlberg and Edward H Egelman and Marek Basler},

doi = {10.1016/j.cell.2015.01.037},

issn = {1097-4172},

year  = {2015},

date = {2015-02-01},

journal = {Cell},

volume = {160},

number = {5},

pages = {952--962},

abstract = {Bacteria use rapid contraction of a long sheath of the type VI secretion system (T6SS) to deliver effectors into a target cell. Here, we present an atomic-resolution structure of a native contracted Vibrio cholerae sheath determined by cryo-electron microscopy. The sheath subunits, composed of tightly interacting proteins VipA and VipB, assemble into a six-start helix. The helix is stabilized by a core domain assembled from four β strands donated by one VipA and two VipB molecules. The fold of inner and middle layers is conserved between T6SS and phage sheaths. However, the structure of the outer layer is distinct and suggests a mechanism of interaction of the bacterial sheath with an accessory ATPase, ClpV, that facilitates multiple rounds of effector delivery. Our results provide a mechanistic insight into assembly of contractile nanomachines that bacteria and phages use to translocate macromolecules across membranes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25620001,

title = {Structural plasticity of helical nanotubes based on coiled-coil assemblies},

author = {E H Egelman and C Xu and F DiMaio and E Magnotti and C Modlin and X Yu and E Wright and D Baker and V P Conticello},

doi = {10.1016/j.str.2014.12.008},

issn = {1878-4186},

year  = {2015},

date = {2015-02-01},

journal = {Structure},

volume = {23},

number = {2},

pages = {280--289},

abstract = {Numerous instances can be seen in evolution in which protein quaternary structures have diverged while the sequences of the building blocks have remained fairly conserved. However, the path through which such divergence has taken place is usually not known. We have designed two synthetic 29-residue α-helical peptides, based on the coiled-coil structural motif, that spontaneously self-assemble into helical nanotubes in vitro. Using electron cryomicroscopy with a newly available direct electron detection capability, we can achieve near-atomic resolution of these thin structures. We show how conservative changes of only one or two amino acids result in dramatic changes in quaternary structure, in which the assemblies can be switched between two very different forms. This system provides a framework for understanding how small sequence changes in evolution can translate into very large changes in supramolecular structure, a phenomenon that may have significant implications for the de novo design of synthetic peptide assemblies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25342806,

title = {High thermodynamic stability of parametrically designed helical bundles},

author = {Po-Ssu Huang and Gustav Oberdorfer and Chunfu Xu and Xue Y Pei and Brent L Nannenga and Joseph M Rogers and Frank DiMaio and Tamir Gonen and Ben Luisi and David Baker},

doi = {10.1126/science.1257481},

issn = {1095-9203},

year  = {2014},

date = {2014-10-01},

journal = {Science},

volume = {346},

number = {6208},

pages = {481--485},

abstract = {We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil-generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid24841284,

title = {Crystal structure of the transport unit of the autotransporter adhesin involved in diffuse adherence from Escherichia coli},

author = {Iris Gawarzewski and Frank DiMaio and Elisa Winterer and Britta Tschapek and Sander H J Smits and Joachim Jose and Lutz Schmitt},

doi = {10.1016/j.jsb.2014.05.003},

issn = {1095-8657},

year  = {2014},

date = {2014-07-01},

journal = {J Struct Biol},

volume = {187},

number = {1},

pages = {20--29},

abstract = {Several serious gastrointestinal diseases, which are widespread all over the world, are caused by enteropathogenic Escherichia coli. The monomeric autotransporter AIDA-I (adhesin involved in diffuse adherence) represents an important virulence factor of these strains and is involved in adhesion, biofilm formation, aggregation and invasion into host cells. Here, we present the crystal structure of the transport unit of AIDA-I at 3.0Å resolution, which forms a 12-stranded β-barrel harboring the linker domain in its pore. Mutagenesis studies of the C-terminal amino acid demonstrated the great impact of this terminal residue on membrane integration of AIDA-I and passenger translocation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid24613918,

title = {Crystal structure analysis of EstA from Arthrobacter sp. Rue61a--an insight into catalytic promiscuity},

author = {Ulrike Gabriella Wagner and Frank DiMaio and Stephan Kolkenbrock and Susanne Fetzner},

doi = {10.1016/j.febslet.2014.02.045},

issn = {1873-3468},

year  = {2014},

date = {2014-04-01},

journal = {FEBS Lett},

volume = {588},

number = {7},

pages = {1154--1160},

abstract = {In this article we analyze the reasons for catalytic promiscuity of a type VIII esterase with β-lactamase fold and the ability to cleave β-lactams. We compared the structure of this enzyme to those of an esterase of the same type without any lactamase ability, an esterase with moderate lactamase ability, and a class C β-lactamase with similar fold. Our results show that for these enzymes, the difference in the substrate specificity is sterically driven.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid23900763,

title = {One contact for every twelve residues allows robust and accurate topology-level protein structure modeling},

author = {David E Kim and Frank Dimaio and Ray Yu-Ruei Wang and Yifan Song and David Baker},

doi = {10.1002/prot.24374},

issn = {1097-0134},

year  = {2014},

date = {2014-02-01},

journal = {Proteins},

volume = {82 Suppl 2},

number = {0 2},

pages = {208--218},

abstract = {A number of methods have been described for identifying pairs of contacting residues in protein three-dimensional structures, but it is unclear how many contacts are required for accurate structure modeling. The CASP10 assisted contact experiment provided a blind test of contact guided protein structure modeling. We describe the models generated for these contact guided prediction challenges using the Rosetta structure modeling methodology. For nearly all cases, the submitted models had the correct overall topology, and in some cases, they had near atomic-level accuracy; for example the model of the 384 residue homo-oligomeric tetramer (Tc680o) had only 2.9 Å root-mean-square deviation (RMSD) from the crystal structure. Our results suggest that experimental and bioinformatic methods for obtaining contact information may need to generate only one correct contact for every 12 residues in the protein to allow accurate topology level modeling.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid24265211,

title = {Relaxation of backbone bond geometry improves protein energy landscape modeling},

author = {Patrick Conway and Michael D Tyka and Frank DiMaio and David E Konerding and David Baker},

doi = {10.1002/pro.2389},

issn = {1469-896X},

year  = {2014},

date = {2014-01-01},

journal = {Protein Sci},

volume = {23},

number = {1},

pages = {47--55},

abstract = {A key issue in macromolecular structure modeling is the granularity of the molecular representation. A fine-grained representation can approximate the actual structure more accurately, but may require many more degrees of freedom than a coarse-grained representation and hence make conformational search more challenging. We investigate this tradeoff between the accuracy and the size of protein conformational search space for two frequently used representations: one with fixed bond angles and lengths and one that has full flexibility. We performed large-scale explorations of the energy landscapes of 82 protein domains under each model, and find that the introduction of bond angle flexibility significantly increases the average energy gap between native and non-native structures. We also find that incorporating bonded geometry flexibility improves low resolution X-ray crystallographic refinement. These results suggest that backbone bond angle relaxation makes an important contribution to native structure energetics, that current energy functions are sufficiently accurate to capture the energetic gain associated with subtle deformations from chain ideality, and more speculatively, that backbone geometry distortions occur late in protein folding to optimize packing in the native state.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid24189231,

title = {Advances in Rosetta structure prediction for difficult molecular-replacement problems},

author = {Frank DiMaio},

doi = {10.1107/S0907444913023305},

issn = {1399-0047},

year  = {2013},

date = {2013-11-01},

journal = {Acta Crystallogr D Biol Crystallogr},

volume = {69},

number = {Pt 11},

pages = {2202--2208},

abstract = {Recent work has shown the effectiveness of structure-prediction methods in solving difficult molecular-replacement problems. The Rosetta protein structure modeling suite can aid in the solution of difficult molecular-replacement problems using templates from 15 to 25% sequence identity; Rosetta refinement guided by noisy density has consistently led to solved structures where other methods fail. In this paper, an overview of the use of Rosetta for these difficult molecular-replacement problems is provided and new modeling developments that further improve model quality are described. Several variations to the method are introduced that significantly reduce the time needed to generate a model and the sampling required to improve the starting template. The improvements are benchmarked on a set of nine difficult cases and it is shown that this improved method obtains consistently better models in less running time. Finally, strategies for best using Rosetta to solve difficult molecular-replacement problems are presented and future directions for the role of structure-prediction methods in crystallography are discussed.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid24076763,

title = {Improved low-resolution crystallographic refinement with Phenix and Rosetta},

author = {Frank DiMaio and Nathaniel Echols and Jeffrey J Headd and Thomas C Terwilliger and Paul D Adams and David Baker},

doi = {10.1038/nmeth.2648},

issn = {1548-7105},

year  = {2013},

date = {2013-11-01},

journal = {Nat Methods},

volume = {10},

number = {11},

pages = {1102--1104},

abstract = {Refinement of macromolecular structures against low-resolution crystallographic data is limited by the ability of current methods to converge on a structure with realistic geometry. We developed a low-resolution crystallographic refinement method that combines the Rosetta sampling methodology and energy function with reciprocal-space X-ray refinement in Phenix. On a set of difficult low-resolution cases, the method yielded improved model geometry and lower free R factors than alternate refinement methods.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid24035711,

title = {High-resolution comparative modeling with RosettaCM},

author = {Yifan Song and Frank DiMaio and Ray Yu-Ruei Wang and David Kim and Chris Miles and Tj Brunette and James Thompson and David Baker},

doi = {10.1016/j.str.2013.08.005},

issn = {1878-4186},

year  = {2013},

date = {2013-10-01},

journal = {Structure},

volume = {21},

number = {10},

pages = {1735--1742},

abstract = {We describe an improved method for comparative modeling, RosettaCM, which optimizes a physically realistic all-atom energy function over the conformational space defined by homologous structures. Given a set of sequence alignments, RosettaCM assembles topologies by recombining aligned segments in Cartesian space and building unaligned regions de novo in torsion space. The junctions between segments are regularized using a loop closure method combining fragment superposition with gradient-based minimization. The energies of the resulting models are optimized by all-atom refinement, and the most representative low-energy model is selected. The CASP10 experiment suggests that RosettaCM yields models with more accurate side-chain and backbone conformations than other methods when the sequence identity to the templates is greater than ∼15%.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid23801759,

title = {Structure of the C-terminal region of an ERG channel and functional implications},

author = {Tinatin I Brelidze and Elena C Gianulis and Frank DiMaio and Matthew C Trudeau and William N Zagotta},

doi = {10.1073/pnas.1306887110},

issn = {1091-6490},

year  = {2013},

date = {2013-07-01},

journal = {Proc Natl Acad Sci U S A},

volume = {110},

number = {28},

pages = {11648--11653},

abstract = {The human ether-à-go-go-related gene (hERG) encodes a K(+) channel crucial for repolarization of the cardiac action potential. EAG-related gene (ERG) channels contain a C-terminal cyclic nucleotide-binding homology domain coupled to the pore of the channel by a C-linker. Here, we report the structure of the C-linker/cyclic nucleotide-binding homology domain of a mosquito ERG channel at 2.5-Å resolution. The structure reveals that the region expected to form the cyclic nucleotide-binding pocket is negatively charged and is occupied by a short β-strand, referred to as the intrinsic ligand, explaining the lack of direct regulation of ERG channels by cyclic nucleotides. In hERG channels, the intrinsic ligand harbors hereditary mutations associated with long-QT syndrome (LQTS), a potentially lethal cardiac arrhythmia. Mutations in the intrinsic ligand affected hERG channel gating and LQTS mutations abolished hERG currents and altered trafficking of hERG channels, which explains the LQT phenotype. The structure also reveals a dramatically different conformation of the C-linker compared with the structures of the related ether-à-go-go-like K(+) and hyperpolarization-activated cyclic nucleotide-modulated channels, suggesting that the C-linker region may be highly dynamic in the KCNH, hyperpolarization-activated cyclic nucleotide-modulated, and cyclic nucleotide-gated channels.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid23592445,

title = {Cryo-EM model validation using independent map reconstructions},

author = {Frank DiMaio and Junjie Zhang and Wah Chiu and David Baker},

doi = {10.1002/pro.2267},

issn = {1469-896X},

year  = {2013},

date = {2013-06-01},

journal = {Protein Sci},

volume = {22},

number = {6},

pages = {865--868},

abstract = {An increasing number of cryo-electron microscopy (cryo-EM) density maps are being generated with suitable resolution to trace the protein backbone and guide sidechain placement. Generating and evaluating atomic models based on such maps would be greatly facilitated by independent validation metrics for assessing the fit of the models to the data. We describe such a metric based on the fit of atomic models with independent test maps from single particle reconstructions not used in model refinement. The metric provides a means to determine the proper balance between the fit to the density and model energy and stereochemistry during refinement, and is likely to be useful in determining values of model building and refinement metaparameters quite generally.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid23633951,

title = {A refined model of the prototypical Salmonella SPI-1 T3SS basal body reveals the molecular basis for its assembly},

author = {Julien R C Bergeron and Liam J Worrall and Nikolaos G Sgourakis and Frank DiMaio and Richard A Pfuetzner and Heather B Felise and Marija Vuckovic and Angel C Yu and Samuel I Miller and David Baker and Natalie C J Strynadka},

doi = {10.1371/journal.ppat.1003307},

issn = {1553-7374},

year  = {2013},

date = {2013-01-01},

journal = {PLoS Pathog},

volume = {9},

number = {4},

pages = {e1003307},

abstract = {The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a "basal body", a lock-nut structure spanning both bacterial membranes, and a "needle" that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid23451892,

title = {Advances, interactions, and future developments in the CNS, Phenix, and Rosetta structural biology software systems},

author = {Paul D Adams and David Baker and Axel T Brunger and Rhiju Das and Frank DiMaio and Randy J Read and David C Richardson and Jane S Richardson and Thomas C Terwilliger},

doi = {10.1146/annurev-biophys-083012-130253},

issn = {1936-1238},

year  = {2013},

date = {2013-01-01},

journal = {Annu Rev Biophys},

volume = {42},

pages = {265--287},

abstract = {Advances in our understanding of macromolecular structure come from experimental methods, such as X-ray crystallography, and also computational analysis of the growing number of atomic models obtained from such experiments. The later analyses have made it possible to develop powerful tools for structure prediction and optimization in the absence of experimental data. In recent years, a synergy between these computational methods for crystallographic structure determination and structure prediction and optimization has begun to be exploited. We review some of the advances in the algorithms used for crystallographic structure determination in the Phenix and Crystallography & NMR System software packages and describe how methods from ab initio structure prediction and refinement in Rosetta have been applied to challenging crystallographic problems. The prospects for future improvement of these methods are discussed.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid22801504,

title = {The dynamic disulphide relay of quiescin sulphydryl oxidase},

author = {Assaf Alon and Iris Grossman and Yair Gat and Vamsi K Kodali and Frank DiMaio and Tevie Mehlman and Gilad Haran and David Baker and Colin Thorpe and Deborah Fass},

doi = {10.1038/nature11267},

issn = {1476-4687},

year  = {2012},

date = {2012-08-01},

journal = {Nature},

volume = {488},

number = {7411},

pages = {414--418},

abstract = {Protein stability, assembly, localization and regulation often depend on the formation of disulphide crosslinks between cysteine side chains. Enzymes known as sulphydryl oxidases catalyse de novo disulphide formation and initiate intra- and intermolecular dithiol/disulphide relays to deliver the disulphides to substrate proteins. Quiescin sulphydryl oxidase (QSOX) is a unique, multi-domain disulphide catalyst that is localized primarily to the Golgi apparatus and secreted fluids and has attracted attention owing to its overproduction in tumours. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulphide-formation pathways. How disulphides are relayed through the multiple redox-active sites of QSOX and whether there is a functional benefit to concatenating these sites on a single polypeptide are open questions. Here we present the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulphide relay were found more than 40 Å apart in this structure, too far for direct disulphide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulphide hand-off, which showed a 165° domain rotation relative to the original structure, bringing the two active sites within disulphide-bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented here, shows further biochemical features that facilitate disulphide transfer in metazoan orthologues. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of new catalytic relays.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid22418934,

title = {phenix.mr_rosetta: molecular replacement and model rebuilding with Phenix and Rosetta},

author = {Thomas C Terwilliger and Frank Dimaio and Randy J Read and David Baker and Gábor Bunkóczi and Paul D Adams and Ralf W Grosse-Kunstleve and Pavel V Afonine and Nathaniel Echols},

doi = {10.1007/s10969-012-9129-3},

issn = {1570-0267},

year  = {2012},

date = {2012-06-01},

journal = {J Struct Funct Genomics},

volume = {13},

number = {2},

pages = {81--90},

abstract = {The combination of algorithms from the structure-modeling field with those of crystallographic structure determination can broaden the range of templates that are useful for structure determination by the method of molecular replacement. Automated tools in phenix.mr_rosetta simplify the application of these combined approaches by integrating Phenix crystallographic algorithms and Rosetta structure-modeling algorithms and by systematically generating and evaluating models with a combination of these methods. The phenix.mr_rosetta algorithms can be used to automatically determine challenging structures. The approaches used in phenix.mr_rosetta are described along with examples that show roles that structure-modeling can play in molecular replacement.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid22550182,

title = {Structure and stoichiometry of an accessory subunit TRIP8b interaction with hyperpolarization-activated cyclic nucleotide-gated channels},

author = {John R Bankston and Stacey S Camp and Frank DiMaio and Alan S Lewis and Dane M Chetkovich and William N Zagotta},

doi = {10.1073/pnas.1201997109},

issn = {1091-6490},

year  = {2012},

date = {2012-05-01},

journal = {Proc Natl Acad Sci U S A},

volume = {109},

number = {20},

pages = {7899--7904},

abstract = {Ion channels operate in intact tissues as part of large macromolecular complexes that can include cytoskeletal proteins, scaffolding proteins, signaling molecules, and a litany of other molecules. The proteins that make up these complexes can influence the trafficking, localization, and biophysical properties of the channel. TRIP8b (tetratricopetide repeat-containing Rab8b-interacting protein) is a recently discovered accessory subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that contributes to the substantial dendritic localization of HCN channels in many types of neurons. TRIP8b interacts with the carboxyl-terminal region of HCN channels and regulates their cell-surface expression level and cyclic nucleotide dependence. Here we examine the molecular determinants of TRIP8b binding to HCN2 channels. Using a single-molecule fluorescence bleaching method, we found that TRIP8b and HCN2 form an obligate 4:4 complex in intact channels. Fluorescence-detection size-exclusion chromatography and fluorescence anisotropy allowed us to confirm that two different domains in the carboxyl-terminal portion of TRIP8b--the tetratricopepide repeat region and the TRIP8b conserved region--interact with two different regions of the HCN carboxyl-terminal region: the carboxyl-terminal three amino acids (SNL) and the cyclic nucleotide-binding domain, respectively. And finally, using X-ray crystallography, we determined the atomic structure of the tetratricopepide region of TRIP8b in complex with a peptide of the carboxy-terminus of HCN2. Together, these experiments begin to uncover the mechanism for TRIP8b binding and regulation of HCN channels.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid22101816,

title = {High-resolution structure of a retroviral protease folded as a monomer},

author = {Miroslaw Gilski and Maciej Kazmierczyk and Szymon Krzywda and Helena Zábranská and Seth Cooper and Zoran Popović and Firas Khatib and Frank DiMaio and James Thompson and David Baker and Iva Pichová and Mariusz Jaskolski},

doi = {10.1107/S0907444911035943},

issn = {1399-0047},

year  = {2011},

date = {2011-11-01},

journal = {Acta Crystallogr D Biol Crystallogr},

volume = {67},

number = {Pt 11},

pages = {907--914},

abstract = {Mason-Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C(α) deviations are large and the active-site 'DTG' loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections, including AIDS.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid21926992,

title = {Crystal structure of a monomeric retroviral protease solved by protein folding game players},

author = {Firas Khatib and Frank DiMaio and  and  and Seth Cooper and Maciej Kazmierczyk and Miroslaw Gilski and Szymon Krzywda and Helena Zabranska and Iva Pichova and James Thompson and Zoran Popović and Mariusz Jaskolski and David Baker},

doi = {10.1038/nsmb.2119},

issn = {1545-9985},

year  = {2011},

date = {2011-09-01},

journal = {Nat Struct Mol Biol},

volume = {18},

number = {10},

pages = {1175--1177},

abstract = {Following the failure of a wide range of attempts to solve the crystal structure of M-PMV retroviral protease by molecular replacement, we challenged players of the protein folding game Foldit to produce accurate models of the protein. Remarkably, Foldit players were able to generate models of sufficient quality for successful molecular replacement and subsequent structure determination. The refined structure provides new insights for the design of antiretroviral drugs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid21873236,

title = {Crystal structure of Toll-like receptor adaptor MAL/TIRAP reveals the molecular basis for signal transduction and disease protection},

author = {Eugene Valkov and Anna Stamp and Frank Dimaio and David Baker and Brett Verstak and Pietro Roversi and Stuart Kellie and Matthew J Sweet and Ashley Mansell and Nicholas J Gay and Jennifer L Martin and Bostjan Kobe},

doi = {10.1073/pnas.1104780108},

issn = {1091-6490},

year  = {2011},

date = {2011-09-01},

journal = {Proc Natl Acad Sci U S A},

volume = {108},

number = {36},

pages = {14879--14884},

abstract = {Initiation of the innate immune response requires agonist recognition by pathogen-recognition receptors such as the Toll-like receptors (TLRs). Toll/interleukin-1 receptor (TIR) domain-containing adaptors are critical in orchestrating the signal transduction pathways after TLR and interleukin-1 receptor activation. Myeloid differentiation primary response gene 88 (MyD88) adaptor-like (MAL)/TIR domain-containing adaptor protein (TIRAP) is involved in bridging MyD88 to TLR2 and TLR4 in response to bacterial infection. Genetic studies have associated a number of unique single-nucleotide polymorphisms in MAL with protection against invasive microbial infection, but a molecular understanding has been hampered by a lack of structural information. The present study describes the crystal structure of MAL TIR domain. Significant structural differences exist in the overall fold of MAL compared with other TIR domain structures: A sequence motif comprising a β-strand in other TIR domains instead corresponds to a long loop, placing the functionally important "BB loop" proline motif in a unique surface position in MAL. The structure suggests possible dimerization and MyD88-interacting interfaces, and we confirm the key interface residues by coimmunoprecipitation using site-directed mutants. Jointly, our results provide a molecular and structural basis for the role of MAL in TLR signaling and disease protection.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid21565698,

title = {Cryo-EM structure of a group II chaperonin in the prehydrolysis ATP-bound state leading to lid closure},

author = {Junjie Zhang and Boxue Ma and Frank DiMaio and Nicholai R Douglas and Lukasz A Joachimiak and David Baker and Judith Frydman and Michael Levitt and Wah Chiu},

doi = {10.1016/j.str.2011.03.005},

issn = {1878-4186},

year  = {2011},

date = {2011-05-01},

journal = {Structure},

volume = {19},

number = {5},

pages = {633--639},

abstract = {Chaperonins are large ATP-driven molecular machines that mediate cellular protein folding. Group II chaperonins use their "built-in lid" to close their central folding chamber. Here we report the structure of an archaeal group II chaperonin in its prehydrolysis ATP-bound state at subnanometer resolution using single particle cryo-electron microscopy (cryo-EM). Structural comparison of Mm-cpn in ATP-free, ATP-bound, and ATP-hydrolysis states reveals that ATP binding alone causes the chaperonin to close slightly with a ∼45° counterclockwise rotation of the apical domain. The subsequent ATP hydrolysis drives each subunit to rock toward the folding chamber and to close the lid completely. These motions are attributable to the local interactions of specific active site residues with the nucleotide, the tight couplings between the apical and intermediate domains within the subunit, and the aligned interactions between two subunits across the rings. This mechanism of structural changes in response to ATP is entirely different from those found in group I chaperonins.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid21543876,

title = {Cloning, expression, purification, crystallization and preliminary X-ray diffraction data of the Pyrococcus horikoshii RadA intein},

author = {Andrzej Lyskowski and Jesper S Oeemig and Anniina Jaakkonen and Katariina Rommi and Frank DiMaio and Dongwen Zhou and Tommi Kajander and David Baker and Alexander Wlodawer and Adrian Goldman and Hideo Iwaï},

doi = {10.1107/S1744309111008372},

issn = {1744-3091},

year  = {2011},

date = {2011-05-01},

journal = {Acta Crystallogr Sect F Struct Biol Cryst Commun},

volume = {67},

number = {Pt 5},

pages = {623--626},

abstract = {The RadA intein from the hyperthermophilic archaebacterium Pyrococcus horikoshii was cloned, expressed and purified for subsequent structure determination. The protein crystallized rapidly in several conditions. The best crystals, which diffracted to 1.75 Å resolution, were harvested from drops consisting of 0.1 M HEPES pH 7.5, 3.0 M NaCl and were cryoprotected with Paratone-N before flash-cooling. The collected data were processed in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 58.1, b = 67.4, c = 82.9 Å. Molecular replacement with Rosetta using energy- and density-guided structure optimization provided the initial solution, which is currently under refinement.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid21532589,

title = {Improved molecular replacement by density- and energy-guided protein structure optimization},

author = {Frank DiMaio and Thomas C Terwilliger and Randy J Read and Alexander Wlodawer and Gustav Oberdorfer and Ulrike Wagner and Eugene Valkov and Assaf Alon and Deborah Fass and Herbert L Axelrod and Debanu Das and Sergey M Vorobiev and Hideo Iwaï and P Raj Pokkuluri and David Baker},

doi = {10.1038/nature09964},

issn = {1476-4687},

year  = {2011},

date = {2011-05-01},

journal = {Nature},

volume = {473},

number = {7348},

pages = {540--543},

abstract = {Molecular replacement procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods, have allowed the rapid solution of large numbers of protein crystal structures. Despite extensive work, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modelling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modelling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction data sets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate that the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction data sets of better than 3.2 Å resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid21466200,

title = {Determination of the structures of symmetric protein oligomers from NMR chemical shifts and residual dipolar couplings},

author = {Nikolaos G Sgourakis and Oliver F Lange and Frank DiMaio and Ingemar André and Nicholas C Fitzkee and Paolo Rossi and Gaetano T Montelione and Ad Bax and David Baker},

doi = {10.1021/ja111318m},

issn = {1520-5126},

year  = {2011},

date = {2011-04-01},

journal = {J Am Chem Soc},

volume = {133},

number = {16},

pages = {6288--6298},

abstract = {Symmetric protein dimers, trimers, and higher-order cyclic oligomers play key roles in many biological processes. However, structural studies of oligomeric systems by solution NMR can be difficult due to slow tumbling of the system and the difficulty in identifying NOE interactions across protein interfaces. Here, we present an automated method (RosettaOligomers) for determining the solution structures of oligomeric systems using only chemical shifts, sparse NOEs, and domain orientation restraints from residual dipolar couplings (RDCs) without a need for a previously determined structure of the monomeric subunit. The method integrates previously developed Rosetta protocols for solving the structures of monomeric proteins using sparse NMR data and for predicting the structures of both nonintertwined and intertwined symmetric oligomers. We illustrated the performance of the method using a benchmark set of nine protein dimers, one trimer, and one tetramer with available experimental data and various interface topologies. The final converged structures are found to be in good agreement with both experimental data and previously published high-resolution structures. The new approach is more readily applicable to large oligomeric systems than conventional structure-determination protocols, which often require a large number of NOEs, and will likely become increasingly relevant as more high-molecular weight systems are studied by NMR.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid21258323,

title = {Crystal structure of XMRV protease differs from the structures of other retropepsins},

author = {Mi Li and Frank Dimaio and Dongwen Zhou and Alla Gustchina and Jacek Lubkowski and Zbigniew Dauter and David Baker and Alexander Wlodawer},

doi = {10.1038/nsmb.1964},

issn = {1545-9985},

year  = {2011},

date = {2011-02-01},

journal = {Nat Struct Mol Biol},

volume = {18},

number = {2},

pages = {227--229},

abstract = {Using energy and density guided Rosetta refinement to improve molecular replacement, we determined the crystal structure of the protease encoded by xenotropic murine leukemia virus-related virus (XMRV). Despite overall similarity of XMRV protease to other retropepsins, the topology of its dimer interface more closely resembles those of the monomeric, pepsin-like enzymes. Thus, XMRV protease may represent a distinct branch of the aspartic protease family.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid21731614,

title = {Modeling symmetric macromolecular structures in Rosetta3},

author = {Frank DiMaio and Andrew Leaver-Fay and Phil Bradley and David Baker and Ingemar André},

doi = {10.1371/journal.pone.0020450},

issn = {1932-6203},

year  = {2011},

date = {2011-01-01},

journal = {PLoS One},

volume = {6},

number = {6},

pages = {e20450},

abstract = {Symmetric protein assemblies play important roles in many biochemical processes. However, the large size of such systems is challenging for traditional structure modeling methods. This paper describes the implementation of a general framework for modeling arbitrary symmetric systems in Rosetta3. We describe the various types of symmetries relevant to the study of protein structure that may be modeled using Rosetta's symmetric framework. We then describe how this symmetric framework is efficiently implemented within Rosetta, which restricts the conformational search space by sampling only symmetric degrees of freedom, and explicitly simulates only a subset of the interacting monomers. Finally, we describe structure prediction and design applications that utilize the Rosetta3 symmetric modeling capabilities, and provide a guide to running simulations on symmetric systems.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid21220301,

title = {Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus},

author = {Dong-Hua Chen and Matthew L Baker and Corey F Hryc and Frank DiMaio and Joanita Jakana and Weimin Wu and Matthew Dougherty and Cameron Haase-Pettingell and Michael F Schmid and Wen Jiang and David Baker and Jonathan A King and Wah Chiu},

doi = {10.1073/pnas.1015739108},

issn = {1091-6490},

year  = {2011},

date = {2011-01-01},

journal = {Proc Natl Acad Sci U S A},

volume = {108},

number = {4},

pages = {1355--1360},

abstract = {Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8- and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T = 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid21073878,

title = {Alternate states of proteins revealed by detailed energy landscape mapping},

author = {Michael D Tyka and Daniel A Keedy and Ingemar André and Frank Dimaio and Yifan Song and David C Richardson and Jane S Richardson and David Baker},

doi = {10.1016/j.jmb.2010.11.008},

issn = {1089-8638},

year  = {2011},

date = {2011-01-01},

journal = {J Mol Biol},

volume = {405},

number = {2},

pages = {607--618},

abstract = {What conformations do protein molecules populate in solution? Crystallography provides a high-resolution description of protein structure in the crystal environment, while NMR describes structure in solution but using less data. NMR structures display more variability, but is this because crystal contacts are absent or because of fewer data constraints? Here we report unexpected insight into this issue obtained through analysis of detailed protein energy landscapes generated by large-scale, native-enhanced sampling of conformational space with Rosetta@home for 111 protein domains. In the absence of tightly associating binding partners or ligands, the lowest-energy Rosetta models were nearly all <2.5 Å C(α)RMSD from the experimental structure; this result demonstrates that structure prediction accuracy for globular proteins is limited mainly by the ability to sample close to the native structure. While the lowest-energy models are similar to deposited structures, they are not identical; the largest deviations are most often in regions involved in ligand, quaternary, or crystal contacts. For ligand binding proteins, the low energy models may resemble the apo structures, and for oligomeric proteins, the monomeric assembly intermediates. The deviations between the low energy models and crystal structures largely disappear when landscapes are computed in the context of the crystal lattice or multimer. The computed low-energy ensembles, with tight crystal-structure-like packing in the core, but more NMR-structure-like variability in loops, may in some cases resemble the native state ensembles of proteins better than individual crystal or NMR structures, and can suggest experimentally testable hypotheses relating alternative states and structural heterogeneity to function.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid20888467,

title = {Analyses of subnanometer resolution cryo-EM density maps},

author = {Matthew L Baker and Mariah R Baker and Corey F Hryc and Frank Dimaio},

doi = {10.1016/S0076-6879(10)83001-0},

issn = {1557-7988},

year  = {2010},

date = {2010-01-01},

journal = {Methods Enzymol},

volume = {483},

pages = {1--29},

abstract = {Today, electron cryomicroscopy (cryo-EM) can routinely achieve subnanometer resolutions of complex macromolecular assemblies. From a density map, one can extract key structural and functional information using a variety of computational analysis tools. At subnanometer resolution, these tools make it possible to isolate individual subunits, identify secondary structures, and accurately fit atomic models. With several cryo-EM studies achieving resolutions beyond 5Å, computational modeling and feature recognition tools have been employed to construct backbone and atomic models of the protein components directly from a density map. In this chapter, we describe several common classes of computational tools that can be used to analyze and model subnanometer resolution reconstructions from cryo-EM. A general protocol for analyzing subnanometer resolution density maps is presented along with a full description of steps used in analyzing the 4.3Å resolution structure of Mm-cpn.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid19596339,

title = {Refinement of protein structures into low-resolution density maps using rosetta},

author = {Frank DiMaio and Michael D Tyka and Matthew L Baker and Wah Chiu and David Baker},

doi = {10.1016/j.jmb.2009.07.008},

issn = {1089-8638},

year  = {2009},

date = {2009-09-01},

journal = {J Mol Biol},

volume = {392},

number = {1},

pages = {181--190},

abstract = {We describe a method based on Rosetta structure refinement for generating high-resolution, all-atom protein models from electron cryomicroscopy density maps. A local measure of the fit of a model to the density is used to directly guide structure refinement and to identify regions incompatible with the density that are then targeted for extensive rebuilding. Over a range of test cases using both simulated and experimentally generated data, the method consistently increases the accuracy of starting models generated either by comparative modeling or by hand-tracing the density. The method can achieve near-atomic resolution starting from density maps at 4-6 A resolution.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid19701941,

title = {Structure prediction for CASP8 with all-atom refinement using Rosetta},

author = {Srivatsan Raman and Robert Vernon and James Thompson and Michael Tyka and Ruslan Sadreyev and Jimin Pei and David Kim and Elizabeth Kellogg and Frank DiMaio and Oliver Lange and Lisa Kinch and Will Sheffler and Bong-Hyun Kim and Rhiju Das and Nick V Grishin and David Baker},

doi = {10.1002/prot.22540},

issn = {1097-0134},

year  = {2009},

date = {2009-01-01},

journal = {Proteins},

volume = {77 Suppl 9},

number = {0 9},

pages = {89--99},

abstract = {We describe predictions made using the Rosetta structure prediction methodology for the Eighth Critical Assessment of Techniques for Protein Structure Prediction. Aggressive sampling and all-atom refinement were carried out for nearly all targets. A combination of alignment methodologies was used to generate starting models from a range of templates, and the models were then subjected to Rosetta all atom refinement. For the 64 domains with readily identified templates, the best submitted model was better than the best alignment to the best template in the Protein Data Bank for 24 cases, and improved over the best starting model for 43 cases. For 13 targets where only very distant sequence relationships to proteins of known structure were detected, models were generated using the Rosetta de novo structure prediction methodology followed by all-atom refinement; in several cases the submitted models were better than those based on the available templates. Of the 12 refinement challenges, the best submitted model improved on the starting model in seven cases. These improvements over the starting template-based models and refinement tests demonstrate the power of Rosetta structure refinement in improving model accuracy.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid19517990,

title = {Spherical-harmonic decomposition for molecular recognition in electron-density maps},

author = {Frank P DiMaio and Ameet B Soni and George N Phillips and Jude W Shavlik},

doi = {10.1504/ijdmb.2009.024852},

issn = {1748-5673},

year  = {2009},

date = {2009-01-01},

journal = {Int J Data Min Bioinform},

volume = {3},

number = {2},

pages = {205--227},

abstract = {Several methods for automatically constructing a protein model from an electron-density map require searching for many small protein-fragment templates in the density. We propose to use the spherical-harmonic decomposition of the template and the maps density to speed this matching. Unlike other template-matching approaches, this allows us to eliminate large portions of the map unlikely to match any templates. We train several first-pass filters for this elimination task. We show our new template-matching method improves accuracy and reduces running time, compared to previous approaches. Finally, we extend our method to produce a structural-homology detection algorithm using electron density.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid17933855,

title = {Creating protein models from electron-density maps using particle-filtering methods},

author = {Frank DiMaio and Dmitry A Kondrashov and Eduard Bitto and Ameet Soni and Craig A Bingman and George N Phillips and Jude W Shavlik},

doi = {10.1093/bioinformatics/btm480},

issn = {1367-4811},

year  = {2007},

date = {2007-11-01},

journal = {Bioinformatics},

volume = {23},

number = {21},

pages = {2851--2858},

abstract = {MOTIVATION: One bottleneck in high-throughput protein crystallography is interpreting an electron-density map, that is, fitting a molecular model to the 3D picture crystallography produces. Previously, we developed ACMI (Automatic Crystallographic Map Interpreter), an algorithm that uses a probabilistic model to infer an accurate protein backbone layout. Here, we use a sampling method known as particle filtering to produce a set of all-atom protein models. We use the output of ACMI to guide the particle filter's sampling, producing an accurate, physically feasible set of structures.nnRESULTS: We test our algorithm on 10 poor-quality experimental density maps. We show that particle filtering produces accurate all-atom models, resulting in fewer chains, lower sidechain RMS error and reduced R factor, compared to simply placing the best-matching sidechains on ACMI's trace. We show that our approach produces a more accurate model than three leading methods--Textal, Resolve and ARP/WARP--in terms of main chain completeness, sidechain identification and crystallographic R factor.nnAVAILABILITY: Source code and experimental density maps available at http://ftp.cs.wisc.edu/machine-learning/shavlik-group/programs/acmi/},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid16873525,

title = {A probabilistic approach to protein backbone tracing in electron density maps},

author = {Frank DiMaio and Jude Shavlik and George N Phillips},

doi = {10.1093/bioinformatics/btl252},

issn = {1367-4811},

year  = {2006},

date = {2006-07-01},

journal = {Bioinformatics},

volume = {22},

number = {14},

pages = {e81--e89},

abstract = {One particularly time-consuming step in protein crystallography is interpreting the electron density map; that is, fitting a complete molecular model of the protein into a 3D image of the protein produced by the crystallographic process. In poor-quality electron density maps, the interpretation may require a significant amount of a crystallographer's time. Our work investigates automating the time-consuming initial backbone trace in poor-quality density maps. We describe ACMI (Automatic Crystallographic Map Interpreter), which uses a probabilistic model known as a Markov field to represent the protein. Residues of the protein are modeled as nodes in a graph, while edges model pairwise structural interactions. Modeling the protein in this manner allows the model to be flexible, considering an almost infinite number of possible conformations, while rejecting any that are physically impossible. Using an efficient algorithm for approximate inference--belief propagation--allows the most probable trace of the protein's backbone through the density map to be determined. We test ACMI on a set of ten protein density maps (at 2.5 to 4.0 A resolution), and compare our results to alternative approaches. At these resolutions, ACMI offers a more accurate backbone trace than current approaches.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}