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

Two structure determination methods, based on the molecular dynamics flexible fitting (MDFF) paradigm, are presented that resolve sub-5-angstrom 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 similar to 25 angstrom demonstrated with the accurate modeling of beta-galactosidase and TRPV1 proteins at 3.2 angstrom and 3.4 angstrom 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.