A Multi-model Approach to Assessing Local and Global Cryo-EM Map Quality.

There does not currently exist a standardized indicator of how well cryo-EM-derived models represent the density from which they were generated. We present a straightforward methodology that utilizes freely available tools to generate a suite of independent models and to evaluate their convergence in an EM density. These analyses provide both a quantitative and qualitative assessment of the precision of the models and their representation of the density, respectively, while concurrently providing a platform for assessing both global and local EM map quality. We further use standardized datasets to provide an expected deviation within a suite of models refined against EM maps reported to be at 5 Å resolution or better. Associating multiple atomic models with a deposited EM map provides a rapid and accessible reporter of convergence, a strong indicator of highly resolved molecular detail, and is an important step toward an FSC-independent assessment of map and model quality.

[1]  Jeroen R Mesters,et al.  An ensemble of crystallographic models enables the description of novel bromate-oxoanion species trapped within a protein crystal. , 2006, Acta crystallographica. Section D, Biological crystallography.

[2]  Sjors H. W. Scheres,et al.  Unravelling biological macromolecules with cryo-electron microscopy , 2016, Nature.

[3]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[4]  Ardan Patwardhan,et al.  EMPIAR: a public archive for raw electron microscopy image data , 2016, Nature Methods.

[5]  Nicholas Furnham,et al.  Conformer generation under restraints. , 2006, Current opinion in structural biology.

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

[7]  Sjors H.W. Scheres,et al.  RELION: Implementation of a Bayesian approach to cryo-EM structure determination , 2012, Journal of structural biology.

[8]  M. Karplus,et al.  Dynamics of folded proteins , 1977, Nature.

[9]  M. Baker,et al.  Outcome of the First Electron Microscopy Validation Task Force Meeting , 2012, Structure.

[10]  Henry van den Bedem,et al.  Exposing Hidden Alternative Backbone Conformations in X-ray Crystallography Using qFit , 2015, bioRxiv.

[11]  M. DePristo,et al.  Heterogeneity and inaccuracy in protein structures solved by X-ray crystallography. , 2004, Structure.

[12]  Wah Chiu,et al.  Near-atomic-resolution cryo-EM for molecular virology. , 2011, Current opinion in virology.

[13]  George Harauz,et al.  Resolution criteria for three dimensional reconstruction , 1986 .

[14]  Kai Zhang,et al.  The structure of the dynactin complex and its interaction with dynein , 2015, Science.

[15]  David Baker,et al.  Cryo‐EM model validation using independent map reconstructions , 2013, Protein science : a publication of the Protein Society.

[16]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[17]  Frank DiMaio,et al.  Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta , 2016, bioRxiv.

[18]  P. Wolynes,et al.  The energy landscapes and motions of proteins. , 1991, Science.

[19]  P. Penczek Resolution measures in molecular electron microscopy. , 2010, Methods in enzymology.

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

[21]  A. Steven,et al.  One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions. , 2013, Journal of structural biology.

[22]  K. Wüthrich Protein structure determination in solution by NMR spectroscopy. , 1990, The Journal of biological chemistry.

[23]  Klaus Schulten,et al.  Quantitative Characterization of Domain Motions in Molecular Machines. , 2017, The journal of physical chemistry. B.

[24]  Hstau Y Liao,et al.  Trajectories of the ribosome as a Brownian nanomachine , 2014, Proceedings of the National Academy of Sciences.

[25]  C. Hill,et al.  The 1.9 A structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions. , 2005, Molecules and Cells.

[26]  M. DePristo,et al.  Is one solution good enough? , 2006, Nature Structural &Molecular Biology.

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

[28]  H. V. D. Bedem,et al.  Automated identification of functional dynamic contact networks from X-ray crystallography , 2013 .

[29]  Adam Frost,et al.  Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains , 2015, Science.

[30]  J. L. Smith,et al.  Structural heterogeneity in protein crystals. , 1986, Biochemistry.

[31]  David Baker,et al.  De novo protein structure determination from near-atomic resolution cryo-EM maps , 2015, Nature Methods.

[32]  Andres E Leschziner,et al.  Visualizing flexibility at molecular resolution: analysis of heterogeneity in single-particle electron microscopy reconstructions. , 2007, Annual review of biophysics and biomolecular structure.

[33]  Yigong Shi,et al.  Sampling the conformational space of the catalytic subunit of human γ-secretase , 2015, bioRxiv.

[34]  F. Schotte,et al.  Protein structural dynamics in solution unveiled via 100-ps time-resolved x-ray scattering , 2010, Proceedings of the National Academy of Sciences.

[35]  D. Clark,et al.  Probing stability–activity relationships in the thermophilic proteasome from Thermoplasma acidophilum by random mutagenesis , 2001, Extremophiles.

[36]  Toshihiko Ogura,et al.  Topology representing network enables highly accurate classification of protein images taken by cryo electron-microscope without masking. , 2003, Journal of structural biology.

[37]  Christopher Irving,et al.  Appion: an integrated, database-driven pipeline to facilitate EM image processing. , 2009, Journal of structural biology.

[38]  Klaus Schulten,et al.  Molecular dynamics-based refinement and validation for sub-5 Å cryo-electron microscopy maps , 2016, eLife.

[39]  Rafael C. Bernardi,et al.  Computational Methodologies for Real-Space Structural Refinement of Large Macromolecular Complexes. , 2016, Annual review of biophysics.

[40]  Andreas Martin,et al.  Atomic structure of the 26S proteasome lid reveals the mechanism of deubiquitinase inhibition , 2016, eLife.

[41]  C. Russo,et al.  Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy , 2017, Nature Communications.

[42]  Marius Schmidt,et al.  Protein energy landscapes determined by five-dimensional crystallography , 2013, Acta crystallographica. Section D, Biological crystallography.

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

[44]  Joseph H. Davis,et al.  Addressing preferred specimen orientation in single-particle cryo-EM through tilting , 2017, Nature Methods.

[45]  E. Nogales,et al.  The proteasome under the microscope: the regulatory particle in focus. , 2013, Current opinion in structural biology.

[46]  H. Ng,et al.  Automated electron‐density sampling reveals widespread conformational polymorphism in proteins , 2010, Protein science : a publication of the Protein Society.

[47]  Alan Brown,et al.  Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions , 2015, Acta crystallographica. Section D, Biological crystallography.

[48]  M. Washburn,et al.  Mediator structure and rearrangements required for holoenzyme formation , 2017, Nature.

[49]  Sriram Subramaniam,et al.  Resolution advances in cryo-EM enable application to drug discovery. , 2016, Current opinion in structural biology.

[50]  Shaoxia Chen,et al.  Prevention of overfitting in cryo-EM structure determination , 2012, Nature Methods.

[51]  R. E. Huber,et al.  High resolution refinement of β‐galactosidase in a new crystal form reveals multiple metal‐binding sites and provides a structural basis for α‐complementation , 2000, Protein science : a publication of the Protein Society.

[52]  Lila M. Gierasch,et al.  Sending Signals Dynamically , 2009, Science.

[53]  B. Alberts The Cell as a Collection of Protein Machines: Preparing the Next Generation of Molecular Biologists , 1998, Cell.

[54]  Sjors H W Scheres,et al.  Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity. , 2015, Molecular cell.

[55]  S. Scheres Beam-induced motion correction for sub-megadalton cryo-EM particles , 2014, eLife.

[56]  G. Lander,et al.  Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex , 2017, Cell.

[57]  W. O. Saxton,et al.  The correlation averaging of a regularly arranged bacterial cell envelope protein , 1982, Journal of microscopy.

[58]  A M Roseman,et al.  FindEM--a fast, efficient program for automatic selection of particles from electron micrographs. , 2004, Journal of structural biology.

[59]  A. Bartesaghi,et al.  2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition , 2016, Science.

[60]  David A Sivak,et al.  E pluribus unum, no more: from one crystal, many conformations. , 2014, Current opinion in structural biology.

[61]  Vincent B. Chen,et al.  Correspondence e-mail: , 2000 .

[62]  George N Phillips,et al.  Ensemble refinement of protein crystal structures: validation and application. , 2007, Structure.

[63]  M Radermacher,et al.  DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy. , 2009, Journal of structural biology.

[64]  F. Sigworth From cryo-EM, multiple protein structures in one shot , 2007, Nature Methods.

[65]  J. Frank,et al.  Structural Basis for Gating and Activation of RyR1 , 2016, Cell.

[66]  Bojan Zagrovic,et al.  X-ray refinement significantly underestimates the level of microscopic heterogeneity in biomolecular crystals , 2014, Nature Communications.

[67]  John L Rubinstein,et al.  Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM , 2015, bioRxiv.

[68]  Yuan He,et al.  Near-atomic resolution visualization of human transcription promoter opening , 2017 .

[69]  N. Gao,et al.  Architecture of the mammalian mechanosensitive Piezo1 channel , 2015, Nature.

[70]  J Bernard Heymann,et al.  Bsoft: image processing and molecular modeling for electron microscopy. , 2007, Journal of structural biology.

[71]  Shirley Coleman,et al.  Validation and Application , 2014 .

[72]  Dmitry Lyumkis,et al.  Modular Assembly of the Bacterial Large Ribosomal Subunit , 2016, Cell.

[73]  Oliver F. Lange,et al.  Recognition Dynamics Up to Microseconds Revealed from an RDC-Derived Ubiquitin Ensemble in Solution , 2008, Science.

[74]  R. Henderson,et al.  High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy☆ , 2013, Ultramicroscopy.

[75]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[76]  Jesse B. Hopkins,et al.  Figures and figure supplements Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography , 2016 .

[77]  Paul D Adams,et al.  Modelling dynamics in protein crystal structures by ensemble refinement , 2012, eLife.

[78]  Randy J. Read,et al.  Interpretation of ensembles created by multiple iterative rebuilding of macromolecular models , 2007, Acta crystallographica. Section D, Biological crystallography.

[79]  R. Henderson,et al.  Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. , 2003, Journal of molecular biology.

[80]  Nathaniel Echols,et al.  EMRinger: Side-chain-directed model and map validation for 3D Electron Cryomicroscopy , 2015, Nature Methods.

[81]  Piotr Neumann,et al.  Validating Resolution Revolution. , 2018, Structure.

[82]  M Karplus,et al.  Effect of anisotropy and anharmonicity on protein crystallographic refinement. An evaluation by molecular dynamics. , 1986, Journal of molecular biology.

[83]  Rama Ranganathan,et al.  Electric-field-stimulated protein mechanics , 2016, Nature.

[84]  Mindy I. Davis,et al.  Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery , 2016, Cell.

[85]  N. Grigorieff,et al.  CTFFIND4: Fast and accurate defocus estimation from electron micrographs , 2015, bioRxiv.

[86]  Sriram Subramaniam,et al.  Structure of β-galactosidase at 3.2-Å resolution obtained by cryo-electron microscopy , 2014, Proceedings of the National Academy of Sciences.

[87]  N. Grigorieff,et al.  Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome , 2016, eLife.

[88]  Conrad C. Huang,et al.  Visualizing density maps with UCSF Chimera. , 2007, Journal of structural biology.

[89]  Chris Wood,et al.  Refinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment , 2016, Methods.

[90]  Pawel A Penczek,et al.  Three-dimensional spectral signal-to-noise ratio for a class of reconstruction algorithms. , 2002, Journal of structural biology.