De Novo modeling in cryo-EM density maps with Pathwalking.

As electron cryo-microscopy (cryo-EM) can now frequently achieve near atomic resolution, accurate interpretation of these density maps in terms of atomistic detail has become paramount in deciphering macromolecular structure and function. However, there are few software tools for modeling protein structure from cryo-EM density maps in this resolution range. Here, we present an extension of our original Pathwalking protocol, which can automatically trace a protein backbone directly from a near-atomic resolution (3-6Å) density map. The original Pathwalking approach utilized a Traveling Salesman Problem solver for backbone tracing, but manual adjustment was still required during modeling. In the new version, human intervention is minimized and we provide a more robust approach for backbone modeling. This includes iterative secondary structure identification, termini detection and the ability to model multiple subunits without prior segmentation. Overall, the new Pathwalking procedure provides a more complete and robust tool for annotating protein structure function in near-atomic resolution density maps.

[1]  Alan Brown,et al.  Structure of the Yeast Mitochondrial Large Ribosomal Subunit , 2014, Science.

[2]  Robert J Collier,et al.  Atomic structure of anthrax protective antigen pore elucidates toxin translocation , 2015 .

[3]  Wen Jiang,et al.  Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo-EM and modeling , 2013, Proceedings of the National Academy of Sciences.

[4]  M. Baker,et al.  Subnanometer-resolution electron cryomicroscopy-based domain models for the cytoplasmic region of skeletal muscle RyR channel , 2008, Proceedings of the National Academy of Sciences.

[5]  Andreas Engel,et al.  Structural determinants of water permeation through aquaporin-1 , 2000, Nature.

[6]  Matthew L. Baker,et al.  Backbone structure of the infectious ε15 virus capsid revealed by electron cryomicroscopy , 2008, Nature.

[7]  M. Baker,et al.  Refinement of protein structures by iterative comparative modeling and CryoEM density fitting. , 2006, Journal of molecular biology.

[8]  Z. Zhou,et al.  3.88 Å structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy , 2008, Nature.

[9]  Matthew L. Baker,et al.  Gorgon and pathwalking: macromolecular modeling tools for subnanometer resolution density maps. , 2012, Biopolymers.

[10]  Zeyun Yu,et al.  Automated segmentation of molecular subunits in electron cryomicroscopy density maps. , 2006, Journal of structural biology.

[11]  Mallur S. Madhusudhan,et al.  CLICK—topology-independent comparison of biomolecular 3D structures , 2011, Nucleic Acids Res..

[12]  A. Cheng,et al.  2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy , 2015, eLife.

[13]  M. Baker,et al.  Protruding knob-like proteins violate local symmetries in an icosahedral marine virus , 2014, Nature Communications.

[14]  M. Baker,et al.  4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus , 2011, The EMBO journal.

[15]  F. Quiocho,et al.  Architecture of the herpes simplex virus major capsid protein derived from structural bioinformatics. , 2003, Journal of molecular biology.

[16]  D. Baker,et al.  Refinement of protein structures into low-resolution density maps using rosetta. , 2009, Journal of molecular biology.

[17]  Marina V. Rodnina,et al.  Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM , 2015, Nature.

[18]  Dong-Hua Chen,et al.  De novo backbone trace of GroEL from single particle electron cryomicroscopy. , 2008, Structure.

[19]  D. Julius,et al.  Structure of the TRPV1 ion channel determined by electron cryo-microscopy , 2013, Nature.

[20]  M. Levitt,et al.  Mechanism of Folding Chamber Closure in a Group II Chaperonin , 2010, Nature.

[21]  Karl Frank,et al.  Structure-Based Characterization of Multiprotein Complexes , 2014, Structure.

[22]  Matthew L. Baker,et al.  An atomic model of brome mosaic virus using direct electron detection and real-space optimization , 2014, Nature Communications.

[23]  F. Dimaio,et al.  Analyses of subnanometer resolution cryo-EM density maps. , 2010, Methods in enzymology.

[24]  William J. Cook,et al.  The Traveling Salesman Problem: A Computational Study , 2007 .

[25]  M. Baker,et al.  Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions , 2003, Nature Structural Biology.

[26]  S. Harrison,et al.  Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction , 2008, Proceedings of the National Academy of Sciences.

[27]  Keld Helsgaun,et al.  General k-opt submoves for the Lin–Kernighan TSP heuristic , 2009, Math. Program. Comput..

[28]  Wen Jiang,et al.  EMAN2: an extensible image processing suite for electron microscopy. , 2007, Journal of structural biology.

[29]  W. Chiu,et al.  Comparison of Segger and other methods for segmentation and rigid-body docking of molecular components in cryo-EM density maps. , 2012, Biopolymers.

[30]  Matthew L. Baker,et al.  Electron cryomicroscopy and bioinformatics suggest protein fold models for rice dwarf virus , 2001, Nature Structural Biology.

[31]  C. Yang,et al.  Cryo-EM structure of a transcribing cypovirus , 2012, Proceedings of the National Academy of Sciences.

[32]  Nicholas Furnham,et al.  Structure of an Xrcc4-DNA ligase IV yeast ortholog complex reveals a novel BRCT interaction mode. , 2006, DNA repair.

[33]  A. Bartesaghi,et al.  2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor , 2015, Science.

[34]  Wah Chiu,et al.  Cryo-EM of macromolecular assemblies at near-atomic resolution , 2010, Nature Protocols.

[35]  M. Baker,et al.  Electron cryomicroscopy of biological machines at subnanometer resolution. , 2005, Structure.

[36]  Wei Zhang,et al.  Combining X-Ray Crystallography and Electron Microscopy , 2005, Structure.

[37]  Yifan Cheng.  Single-Particle Cryo-EM at Crystallographic Resolution , 2015, Cell.

[38]  Keren Lasker,et al.  Finding the right fit: chiseling structures out of cryo-electron microscopy maps. , 2014, Current opinion in structural biology.

[39]  M. Baker,et al.  Bridging the information gap: computational tools for intermediate resolution structure interpretation. , 2001, Journal of molecular biology.

[40]  Wah Chiu,et al.  Constructing and validating initial Cα models from subnanometer resolution density maps with pathwalking. , 2012, Structure.

[41]  Bradley L. Pentelute,et al.  Atomic structure of anthrax PA pore elucidates toxin translocation , 2015, Nature.

[42]  D. Agard,et al.  Electron counting and beam-induced motion correction enable near atomic resolution single particle cryoEM , 2013, Nature Methods.

[43]  Daisuke Kihara,et al.  Computational methods for constructing protein structure models from 3D electron microscopy maps. , 2013, Journal of structural biology.

[44]  D. Julius,et al.  TRPV1 structures in distinct conformations reveal mechanisms of activation , 2013, Nature.

[45]  M. Baker,et al.  Structural characterization of components of protein assemblies by comparative modeling and electron cryo-microscopy. , 2005, Journal of structural biology.

[46]  Matthew L. Baker,et al.  Structural Changes in a Marine Podovirus Associated with Release of its Genome into Prochlorococcus , 2010, Nature Structural &Molecular Biology.

[47]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[48]  B. Honig,et al.  Building and refining protein models within cryo-electron microscopy density maps based on homology modeling and multiscale structure refinement. , 2010, Journal of molecular biology.

[49]  M. Baker,et al.  Identification of secondary structure elements in intermediate-resolution density maps. , 2007, Structure.

[50]  M. Baker,et al.  Gating machinery of InsP3R channels revealed by electron cryomicroscopy , 2015, Nature.