Cryoem-Guided Iterative Molecular Dynamics - Rosetta Protein Structure Refinement Protocol Improves Protein Model Quality

Abstract

Having reliable computational tools for predicting high-resolution protein structures from medium resolution cryoEM density maps becomes more and more important in light of the recent rise in sub-nanometer resolution cryoEM density maps. While programs like EM-Fold, Rosetta or SSEBuilder can accurately predict protein models correct at the topology level - secondary structure elements will be in the correct positions of the density map -, the refinement of those models to atomic resolution remains a challenge in the field. Previously, we had presented an iterative Rosetta - Molecular Dynamics (MD) protocol, showing that iteration of Rosetta with an orthogonal sampling and scoring strategy can facilitate exploration of conformational space and build atomic resolution protein models. Here we present an improved iterative protein refinement protocol which is guided by the cryoEM density map at all stages and does not require previous knowledge of the protein structure. We ran short cryoEM-guided MDFF simulations on models created by de novo folding of large proteins into cryoEM density maps to enable sampling of conformational space not directly accessible to Rosetta all-atom refinement and thus provide an escape route from conformational traps. This cryoEM-guided, combined MD - Rosetta protein structure refinement protocol overcomes some of the sampling limitations that each individual method is subject to. Three of four benchmark proteins showed substantial improvement through three rounds of the iterative refinement protocol. Models with less than 1.5 A RMSD to the native structure were built for proteins with more than 150 residues. Molecular dynamics is most efficient in applying subtle but important rearrangements within secondary structure elements, and is thus highly complementary to the Rosetta refinement which focuses on side chains and loop regions.