Re-evaluation of low-resolution crystal structures via interactive molecular-dynamics flexible fitting (iMDFF): a case study in complement C4.

While the rapid proliferation of high-resolution structures in the Protein Data Bank provides a rich set of templates for starting models, it remains the case that a great many structures both past and present are built at least in part by hand-threading through low-resolution and/or weak electron density. With current model-building tools this task can be challenging, and the de facto standard for acceptable error rates (in the form of atomic clashes and unfavourable backbone and side-chain conformations) in structures based on data with dmax not exceeding 3.5 Å reflects this. When combined with other factors such as model bias, these residual errors can conspire to make more serious errors in the protein fold difficult or impossible to detect. The three recently published 3.6-4.2 Å resolution structures of complement C4 (PDB entries 4fxg, 4fxk and 4xam) rank in the top quartile of structures of comparable resolution both in terms of Rfree and MolProbity score, yet, as shown here, contain register errors in six β-strands. By applying a molecular-dynamics force field that explicitly models interatomic forces and hence excludes most physically impossible conformations, the recently developed interactive molecular-dynamics flexible fitting (iMDFF) approach significantly reduces the complexity of the conformational space to be searched during manual rebuilding. This substantially improves the rate of detection and correction of register errors, and allows user-guided model building in maps with a resolution lower than 3.5 Å to converge to solutions with a stereochemical quality comparable to atomic resolution structures. Here, iMDFF has been used to individually correct and re-refine these three structures to MolProbity scores of <1.7, and strategies for working with such challenging data sets are suggested. Notably, the improved model allowed the resolution for complement C4b to be extended from 4.2 to 3.5 Å as demonstrated by paired refinement.

[1]  Krista Joosten,et al.  PDB_REDO: constructive validation, more than just looking for errors , 2012, Acta crystallographica. Section D, Biological crystallography.

[2]  G. Ambrus,et al.  A True Autoactivating Enzyme , 2005, Journal of Biological Chemistry.

[3]  T. Yeates,et al.  Verification of protein structures: Patterns of nonbonded atomic interactions , 1993, Protein science : a publication of the Protein Society.

[4]  John D Lambris,et al.  Structure of C3b-factor H and implications for host protection by complement regulators , 2009, Nature Immunology.

[5]  Jacob M Litman,et al.  Dead-End Elimination with a Polarizable Force Field Repacks PCNA Structures. , 2015, Biophysical journal.

[6]  Youngdo Won.  Force field for monovalent, divalent, and trivalent cations developed under the solvent boundary potential. , 2012, The journal of physical chemistry. A.

[7]  S. Thiel,et al.  Toward a structure-based comprehension of the lectin pathway of complement. , 2013, Molecular immunology.

[8]  S. A. Law.  THE COVALENT BINDING REACTION OF C3 AND C4 a , 1983, Annals of the New York Academy of Sciences.

[9]  Jing Huang,et al.  CHARMM36 all‐atom additive protein force field: Validation based on comparison to NMR data , 2013, J. Comput. Chem..

[10]  C. Levinthal.  Molecular model-building by computer. , 1966, Scientific American.

[11]  M. Kjeldgaard,et al.  O: A Macromolecule Modeling Environment , 1990 .

[12]  P. Roversi,et al.  Structural basis for complement factor I control and its disease-associated sequence polymorphisms , 2011, Proceedings of the National Academy of Sciences.

[13]  Nathaniel Echols,et al.  Improved low-resolution crystallographic refinement with Phenix and Rosetta , 2013, Nature Methods.

[14]  Brian J. Smith,et al.  Higher-Resolution Structure of the Human Insulin Receptor Ectodomain: Multi-Modal Inclusion of the Insert Domain. , 2016, Structure.

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

[16]  G. Larson,et al.  LC-MS/MS characterization of O-glycosylation sites and glycan structures of human cerebrospinal fluid glycoproteins. , 2013, Journal of proteome research.

[17]  Klaus Schulten,et al.  xMDFF: molecular dynamics flexible fitting of low-resolution X-ray structures , 2014, Acta crystallographica. Section D, Biological crystallography.

[18]  Charles E. Bugg,et al.  Crystallographic and Modeling Methods in Molecular Design , 1990, Springer New York.

[19]  C. Sander,et al.  Errors in protein structures , 1996, Nature.

[20]  V. Ramakrishnan,et al.  X‐ray crystallography shows that translational initiation factor IF3 consists of two compact alpha/beta domains linked by an alpha‐helix. , 1995, The EMBO journal.

[21]  Ian W. Davis,et al.  Structure validation by Cα geometry: ϕ,ψ and Cβ deviation , 2003, Proteins.

[22]  J. Richardson,et al.  The penultimate rotamer library , 2000, Proteins.

[23]  L. Lo Leggio,et al.  The structure and specificity of Escherichia coli maltose acetyltransferase give new insight into the LacA family of acyltransferases. , 2003, Biochemistry.

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

[25]  Narayanan Eswar,et al.  Protein structure modeling with MODELLER. , 2008, Methods in molecular biology.

[26]  J. Jensen,et al.  Solution Structures of Complement C2 and Its C4 Complexes Propose Pathway-specific Mechanisms for Control and Activation of the Complement Proconvertases* , 2016, The Journal of Biological Chemistry.

[27]  Laxmikant V. Kalé,et al.  Scalable molecular dynamics with NAMD , 2005, J. Comput. Chem..

[28]  Abhik Mukhopadhyay,et al.  PDBe: improved accessibility of macromolecular structure data from PDB and EMDB , 2015, Nucleic Acids Res..

[29]  S. Thiel,et al.  Complement activation, regulation, and molecular basis for complement‐related diseases , 2015, The EMBO journal.

[30]  Klaus Schulten,et al.  A system for interactive molecular dynamics simulation , 2001, I3D '01.

[31]  Ericka Stricklin-Parker,et al.  Ann , 2005 .

[32]  C. Y. Yu,et al.  Genetic, structural and functional diversities of human complement components C4A and C4B and their mouse homologues, Slp and C4. , 2001, International immunopharmacology.

[33]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[34]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[35]  Y. Kang,et al.  Positional preference of proline in α‐helices , 1999, Protein science : a publication of the Protein Society.

[36]  N. S. Laursen,et al.  Structural basis for activation of the complement system by component C4 cleavage , 2012, Proceedings of the National Academy of Sciences.

[37]  P. Andrew Karplus,et al.  Linking Crystallographic Model and Data Quality , 2012, Science.

[38]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[39]  Piet Gros,et al.  Structures of complement component C3 provide insights into the function and evolution of immunity , 2005, Nature.

[40]  M. Gadjeva,et al.  New insights into the molecular mechanisms of classical complement activation. , 2010, Molecular immunology.

[41]  I. Rayment,et al.  Trimethyllead acetate: a first-choice heavy atom derivative for protein crystallography. , 1991, Archives of biochemistry and biophysics.

[42]  K. Diederichs,et al.  Better models by discarding data? , 2013, Acta crystallographica. Section D, Biological crystallography.

[43]  D. Turk.  MAIN software for density averaging, model building, structure refinement and validation , 2013, Acta crystallographica. Section D, Biological crystallography.

[44]  Paul D. Adams,et al.  Use of knowledge-based restraints in phenix.refine to improve macromolecular refinement at low resolution , 2012, Acta crystallographica. Section D, Biological crystallography.

[45]  G. Hortin,et al.  Identification of the site of sulfation of the fourth component of human complement. , 1986, The Journal of biological chemistry.

[46]  Fei Long,et al.  The PDB_REDO server for macromolecular structure model optimization , 2014, IUCrJ.

[47]  M. Lawrence,et al.  Structure of the insulin receptor ectodomain reveals a folded-over conformation , 2006, Nature.

[48]  G J Kleywegt,et al.  Model building and refinement practice. , 1997, Methods in enzymology.

[49]  Á. Szilágyi,et al.  Structural Basis for the Function of Complement Component C4 within the Classical and Lectin Pathways of Complement , 2015, The Journal of Immunology.

[50]  P. Zwart,et al.  Towards automated crystallographic structure refinement with phenix.refine , 2012, Acta crystallographica. Section D, Biological crystallography.

[51]  R. Levine,et al.  Covalent binding and hemolytic activity of complement proteins. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[52]  G. Ambrus,et al.  The structure of MBL-associated serine protease-2 reveals that identical substrate specificities of C1s and MASP-2 are realized through different sets of enzyme-substrate interactions. , 2004, Journal of molecular biology.