Iterative elastic 3D-to-2D alignment method using normal modes for studying structural dynamics of large macromolecular complexes.

This article presents a method to study large-scale conformational changes by combining electron microscopy (EM) single-particle image analysis and normal mode analysis (NMA). It is referred to as HEMNMA, which stands for hybrid electron microscopy normal mode analysis. NMA of a reference structure (atomic-resolution structure or EM volume) is used to predict possible motions that are then confronted with EM images within an automatic iterative elastic 3D-to-2D alignment procedure to identify actual motions in the imaged samples. HEMNMA can be used to extensively analyze the conformational changes and may be used in combination with classic discrete procedures. The identified conformations allow modeling of deformation pathways compatible with the experimental data. HEMNMA was tested with synthetic and experimental data sets of E. coli 70S ribosome, DNA polymerase Pol α and B subunit complex of the eukaryotic primosome, and tomato bushy stunt virus.

[1]  J. Witz,et al.  Structural dynamics, an intrinsic property of viral capsids , 2001, Archives of Virology.

[2]  D. Ming,et al.  How to describe protein motion without amino acid sequence and atomic coordinates , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[3]  N Boisset,et al.  Divalent ion-dependent swelling of tomato bushy stunt virus: a multi-approach study. , 2005, Biochimica et biophysica acta.

[4]  Tirion,et al.  Large Amplitude Elastic Motions in Proteins from a Single-Parameter, Atomic Analysis. , 1996, Physical review letters.

[5]  Karsten Suhre,et al.  NORMA: a tool for flexible fitting of high-resolution protein structures into low-resolution electron-microscopy-derived density maps. , 2006, Acta crystallographica. Section D, Biological crystallography.

[6]  F. Tama,et al.  Normal mode based flexible fitting of high-resolution structure into low-resolution experimental data from cryo-EM. , 2004, Journal of structural biology.

[7]  M. J. D. Powell,et al.  UOBYQA: unconstrained optimization by quadratic approximation , 2002, Math. Program..

[8]  F. Asturias,et al.  Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo–electron microscopy , 2006, Nature Structural &Molecular Biology.

[9]  Alberto D. Pascual-Montano,et al.  3DEM Loupe: analysis of macromolecular dynamics using structures from electron microscopy , 2013, Nucleic Acids Res..

[10]  Luca Pellegrini,et al.  3D architecture of DNA Pol α reveals the functional core of multi-subunit replicative polymerases , 2009, The EMBO journal.

[11]  Joachim Frank,et al.  Locking and Unlocking of Ribosomal Motions , 2003, Cell.

[12]  C. Brooks,et al.  Symmetry, form, and shape: guiding principles for robustness in macromolecular machines. , 2006, Annual review of biophysics and biomolecular structure.

[13]  Pawel A Penczek,et al.  Cryo-electron microscopy studies of human TFIID: conformational breathing in the integration of gene regulatory cues. , 2006, Structure.

[14]  M. Delarue,et al.  On the use of low-frequency normal modes to enforce collective movements in refining macromolecular structural models. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Robert E. Johnson,et al.  Structural insights into yeast DNA polymerase delta by small angle X-ray scattering. , 2009, Journal of molecular biology.

[16]  E. Orlova,et al.  Detection and separation of heterogeneity in molecular complexes by statistical analysis of their two-dimensional projections. , 2008, Journal of structural biology.

[17]  R. Jernigan,et al.  Global ribosome motions revealed with elastic network model. , 2004, Journal of structural biology.

[18]  J M Carazo,et al.  XMIPP: a new generation of an open-source image processing package for electron microscopy. , 2004, Journal of structural biology.

[19]  C. Brooks,et al.  Diversity and identity of mechanical properties of icosahedral viral capsids studied with elastic network normal mode analysis. , 2005, Journal of molecular biology.

[20]  G. Herman,et al.  Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization , 2007, Nature Methods.

[21]  W. Wriggers,et al.  Exploring global distortions of biological macromolecules and assemblies from low-resolution structural information and elastic network theory. , 2002, Journal of molecular biology.

[22]  Roberto Melero,et al.  Flexible tethering of primase and DNA Pol α in the eukaryotic primosome , 2011, Nucleic acids research.

[23]  J. Cate,et al.  Structures of the Ribosome in Intermediate States of Ratcheting , 2009, Science.

[24]  F. Tama,et al.  Flexible multi-scale fitting of atomic structures into low-resolution electron density maps with elastic network normal mode analysis. , 2004, Journal of molecular biology.

[25]  S Jonić,et al.  Spline-based image-to-volume registration for three-dimensional electron microscopy. , 2005, Ultramicroscopy.

[26]  J M Carazo,et al.  A multiresolution approach to orientation assignment in 3D electron microscopy of single particles. , 2004, Journal of structural biology.

[27]  J. Trewhella.  X‐Ray Scattering , 2002 .

[28]  J. Frank,et al.  A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation. , 2006, Journal of structural biology.

[29]  Karsten Suhre,et al.  ElNémo: a normal mode web server for protein movement analysis and the generation of templates for molecular replacement , 2004, Nucleic Acids Res..

[30]  Y. Sanejouand,et al.  Conformational change of proteins arising from normal mode calculations. , 2001, Protein engineering.

[31]  Joachim Frank,et al.  Cryoelectron microscopy structures of the ribosome complex in intermediate states during tRNA translocation , 2011, Proceedings of the National Academy of Sciences.

[32]  J Witz,et al.  Divalent ion-dependent reversible swelling of tomato bushy stunt virus and organization of the expanded virion. , 1982, Journal of molecular biology.

[33]  R. Brüschweiler.  Collective protein dynamics and nuclear spin relaxation , 1995 .

[34]  S. Wakil,et al.  Experimental verification of conformational variation of human fatty acid synthase as predicted by normal mode analysis. , 2004, Structure.

[35]  Sergei L. Dudarev,et al.  Robust Parameterization of Elastic and Absorptive Electron Atomic Scattering Factors , 1996 .

[36]  J. Frank,et al.  Dynamic reorganization of the functionally active ribosome explored by normal mode analysis and cryo-electron microscopy , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[37]  J M Carazo,et al.  A method for estimating the CTF in electron microscopy based on ARMA models and parameter adjustment. , 2003, Ultramicroscopy.

[38]  I. K. Robinson,et al.  Structure of the expanded state of tomato bushy stunt virus , 1982, Nature.

[39]  Bruno P. Klaholz,et al.  Structure of the 30S translation initiation complex , 2008, Nature.

[40]  Florence Tama,et al.  Mega-Dalton biomolecular motion captured from electron microscopy reconstructions. , 2003, Journal of molecular biology.