Molecular dynamics-based refinement and validation for sub-5 Å cryo-electron microscopy maps.

Abhishek Singharoy,Jianhua Zhao,Ryan Mcgreevy,John E Stone,Klaus Schulten,Ivan Teo

doi:10.7554/elife.16105

Abstract

Two structure determination methods, based on the molecular dynamics flexible fitting (MDFF) paradigm, are presented that resolve sub-5 Å cryo-electron microscopy (EM) maps with either single structures or ensembles of such structures. The methods, denoted cascade MDFF and resolution exchange MDFF, sequentially re-refine a search model against a series of maps of progressively higher resolutions, which ends with the original experimental resolution. Application of sequential re-refinement enables MDFF to achieve a radius of convergence of ~25 Å demonstrated with the accurate modeling of β-galactosidase and TRPV1 proteins at 3.2 Å and 3.4 Å resolution, respectively. The MDFF refinements uniquely offer map-model validation and B-factor determination criteria based on the inherent dynamics of the macromolecules studied, captured by means of local root mean square fluctuations. The MDFF tools described are available to researchers through an easy-to-use and cost-effective cloud computing resource on Amazon Web Services.

Highlights

Cryo-electron microscopy has evolved into one of the most effective structure determination tools in modern day structural biology, achieving in recent years resolutions rivalling those of X-ray crystallography or NMR spectroscopy (Cheng, 2015)
We first describe the methodological advances achieved within cascade MDFF (cMDFF) and resolution exchange MDFF (ReMDFF) for the resolution of sub-5 Amaps
Refinement, and structure validation protocols based on these advances are subsequently demonstrated for five exemplary protein complexes that were chosen based on the availability of high-resolution (3–5 A ) electron microscopy (EM) maps and atomic structures

Summary

Introduction

Cryo-electron microscopy (cryo-EM) has evolved into one of the most effective structure determination tools in modern day structural biology, achieving in recent years resolutions rivalling those of X-ray crystallography or NMR spectroscopy (Cheng, 2015). Cryo-EM based structure determination overcomes two major bottlenecks faced in traditional X-ray crystallography, namely, the arduous task of preparing well-ordered crystals of macromolecules (Unger, 2002), and the more fundamental problem with capturing these molecules in unphysiological states as a result of crystal contacts (Neutze et al, 2015). Cryo-EM provides a natural way of resolving the structures of large macromolecular complexes. Various real-space refinement methods that combine crystallographic structures and cryo-EM densities for structure determination have been developed, including DireX (Schroder et al, 2007), Flex-EM (Topf et al, 2008), Rosetta (DiMaio et al, 2015), FRODA (Jolley et al, 2008), Phenix real space refinement (Afonine et al, 2013), and Molecular Dynamics Flexible Fitting (MDFF) (Trabuco et al, 2008, 2009; McGreevy et al, 2016).

Methods

Results

Conclusion