Abstract
The recent rapid development of single-particle electron cryo-microscopy (cryo-EM) now allows structures to be solved by this method at resolutions close to 3 Å. Here, a number of tools to facilitate the interpretation of EM reconstructions with stereochemically reasonable all-atom models are described. The BALBES database has been repurposed as a tool for identifying protein folds from density maps. Modifications to Coot, including new Jiggle Fit and morphing tools and improved handling of nucleic acids, enhance its functionality for interpreting EM maps. REFMAC has been modified for optimal fitting of atomic models into EM maps. As external structural information can enhance the reliability of the derived atomic models, stabilize refinement and reduce overfitting, ProSMART has been extended to generate interatomic distance restraints from nucleic acid reference structures, and a new tool, LIBG, has been developed to generate nucleic acid base-pair and parallel-plane restraints. Furthermore, restraint generation has been integrated with visualization and editing in Coot, and these restraints have been applied to both real-space refinement in Coot and reciprocal-space refinement in REFMAC.
Highlights
Single-particle electron cryo-microscopy is currently undergoing a technical revolution (Kuhlbrandt, 2014; Smith & Rubinstein, 2014)
Single-particle cryo-EM is a rapidly developing technique that is capable of delivering structures at resolutions similar to those achieved by X-ray crystallography
We have presented a number of new tools to facilitate the interpretation of EM maps, from initial density-based fold identification through model building to refinement and validation
Summary
Single-particle electron cryo-microscopy (cryo-EM) is currently undergoing a technical revolution (Kuhlbrandt, 2014; Smith & Rubinstein, 2014). The BALBES pipeline comprises a nonredundant database of approximately 50 000 protein domains greater than 15 amino acids in length and refined against data extending to resolution limits of better than 3.5 A. The structure of mL38 (PDB entry 3j6b, chain 1; Amunts et al, 2014) was used to identify structural homologues in the PDB (Krissinel & Henrick, 2004), with the best match sharing the same fold as 1kn (PDB entry 1wpx; Mima et al, 2005) but resolved at a lower resolution This confirms that the BALBES–MOLREP pipeline identified the best possible solution from over 14 000 domains. To improve the functionality of Coot for EM, we have implemented a number of new tools (detailed below) that are applicable to X-ray crystallography
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