EMBuilder: A Template Matching-based Automatic Model-building Program for High-resolution Cryo-Electron Microscopy Maps

The resolution of electron-potential maps in single-particle cryo-electron microscopy (cryoEM) is approaching atomic or near- atomic resolution. However, no program currently exists for de novo cryoEM model building at resolutions exceeding beyond 3.5 Å. Here, we present a program, EMBuilder, based on template matching, to generate cryoEM models at high resolution. The program identifies features in both secondary-structure and Cα stages. In the secondary structure stage, helices and strands are identified with pre-computed templates, and the voxel size of the entire map is then refined to account for microscopic magnification errors. The identified secondary structures are then extended from both ends in the Cα stage via a log-likelihood (LLK) target function, and if possible, the side chains are also assigned. This program can build models of large proteins (~1 MDa) in a reasonable amount of time (~1 day) and thus has the potential to greatly decrease the manual workload required for model building of high-resolution cryoEM maps.

[1]  Kevin Cowtan,et al.  The Buccaneer software for automated model building , 2006 .

[2]  Sjors H. W. Scheres,et al.  An atomic structure of human γ-secretase , 2015, Nature.

[3]  Richard J Morris,et al.  Towards complete validated models in the next generation of ARP/wARP. , 2004, Acta crystallographica. Section D, Biological crystallography.

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

[5]  M. Rossmann,et al.  Fitting atomic models into electron-microscopy maps. , 2000, Acta crystallographica. Section D, Biological crystallography.

[6]  D. Julius,et al.  TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action , 2016, Nature.

[7]  Irina Massova,et al.  Crystallographic and computational insight on the mechanism of zinc-ion-dependent inactivation of carboxypeptidase a by 2-benzyl-3-iodopropanoate. , 1996 .

[8]  Randy J. Read,et al.  Dauter Iterative model building , structure refinement and density modification with the PHENIX AutoBuild wizard , 2007 .

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

[10]  Prashant Rao,et al.  Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase , 2016, Molecular Pharmacology.

[11]  John D. Westbrook,et al.  EMDataBank unified data resource for 3DEM , 2013, Nucleic Acids Res..

[12]  T. Terwilliger.  Maximum-likelihood density modification using pattern recognition of structural motifs , 2001, Acta crystallographica. Section D, Biological crystallography.

[13]  K Cowtan,et al.  Fast Fourier feature recognition. , 2001, Acta crystallographica. Section D, Biological crystallography.

[14]  Muyuan Chen,et al.  De Novo modeling in cryo-EM density maps with Pathwalking. , 2016, Journal of structural biology.

[15]  P. Stewart,et al.  EM-fold: de novo atomic-detail protein structure determination from medium-resolution density maps. , 2012, Structure.

[16]  W. Kühlbrandt.  The Resolution Revolution , 2014, Science.

[17]  Kevin Cowtan,et al.  Fitting molecular fragments into electron density , 2007, Acta crystallographica. Section D, Biological crystallography.

[18]  J Berendzen,et al.  Crystal structures of myoglobin-ligand complexes at near-atomic resolution. , 1999, Biophysical journal.

[19]  W Chiu,et al.  Tools for Model Building and Optimization into Near-Atomic Resolution Electron Cryo-Microscopy Density Maps , 2016, Methods in Enzymology.

[20]  M. Baker,et al.  Modeling protein structure at near atomic resolutions with Gorgon. , 2011, Journal of structural biology.

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

[22]  M. Bühner,et al.  Studies of Glutamate Dehydrogenase , 1974 .

[23]  Jude W. Shavlik,et al.  Creating protein models from electron-density maps using particle-filtering methods , 2007, Bioinform..

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

[25]  D. Barford,et al.  Molecular mechanism of APC/C activation by mitotic phosphorylation , 2016, Nature.

[26]  M. Rossmann,et al.  Combining electron microscopic with x-ray crystallographic structures. , 2001, Journal of structural biology.

[27]  E B Krissinel,et al.  The new CCP4 Coordinate Library as a toolkit for the design of coordinate-related applications in protein crystallography. , 2004, Acta crystallographica. Section D, Biological crystallography.

[28]  Thomas R Ioerger,et al.  Automatic modeling of protein backbones in electron-density maps via prediction of Calpha coordinates. , 2002, Acta crystallographica. Section D, Biological crystallography.

[29]  Daniel W. Kulp,et al.  Generalized Fragment Picking in Rosetta: Design, Protocols and Applications , 2011, PloS one.

[30]  M. O’Donnell,et al.  Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation , 2016 .

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

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

[33]  Kevin Cowtan,et al.  The Buccaneer software for automated model building. 1. Tracing protein chains. , 2006, Acta crystallographica. Section D, Biological crystallography.

[34]  G. Kleywegt,et al.  Detecting folding motifs and similarities in protein structures. , 1997, Methods in enzymology.