Cryo-EM structure and B-factor refinement with ensemble representation

Abstract

Abstract Cryo-EM experiments produce images of macromolecular assemblies that are combined to produce three-dimensional density maps. Typically, atomic models of the constituent molecules are fitted into these maps, followed by a density-guided refinement. We introduce TEMPy-ReFF, a method for atomic structure refinement in cryo-EM density maps. Our method represents atomic positions as components of a Gaussian mixture model, utilising their variances as B-factors, which are used to derive an ensemble description. Extensively tested on a substantial dataset of 229 cryo-EM maps from EMDB ranging in resolution from 2.1-4.9 Å with corresponding PDB and CERES atomic models, our results demonstrate that TEMPy-ReFF ensembles provide a superior representation of cryo-EM maps. On a single-model basis, it performs similarly to the CERES re-refinement protocol, although there are cases where it provides a better fit to the map. Furthermore, our method enables the creation of composite maps free of boundary artefacts. TEMPy-ReFF is useful for better interpretation of flexible structures, such as those involving RNA, DNA or ligands.