Current approaches for the fitting and refinement of atomic models into cryo-EM maps usingCCP-EM

Abstract

REFMAC5 and related tools for the refinement of atomic models into cryo-EM reconstructions in CCP-EM are reviewed. An upper bound on the correlation between observed and calculated Fourier coefficients is identified, and the practical utility of map blurring/sharpening is discussed. The Divide and Conquer pipeline for refining large complexes in parallel, and ProSHADE for the identification of symmetries in a given map or coordinate set, are presented.

Structure of the far-red light utilizing photosystem I of Acaryochloris

“…Each model of the three monomers in the PSI trimer was re ned separately, and the re ned model was modi ed using COOT 57 manually to t the cryo-EM density map. Subsequently, the obtained structure model was nally re ned using refmac5 58 (Extended Data Fig. 11).…”

Current approaches for automated model building into cryo-EM maps using Buccaneer with CCP-EM

“…This technique has allowed the study of rapidly frozen biological macromolecules without the need for crystallization. The increasing number of cryo-EM maps produced in recent years, especially those of high resolution (<4 Å ), has motivated the modification of existing and the development of new model-building tools to interpret cryo-EM maps (Cowtan, 2008;Baker et al, 2012;Brown et al, 2015;Wang et al, 2015;Chen et al, 2016;Zhou et al, 2017;Terashi & Kihara, 2018;Chojnowski et al, 2018;Nicholls et al, 2018).…”

“…However, using appropriately modified scattering factors allows the use of the existing Fourier-based techniques that were developed for the analysis of X-ray data. Nevertheless, there are qualitative differences that need to be accounted for (Nicholls et al, 2018) and appropriate adjustments have to be made in order for the existing tools to work well with cryo-EM data. In this work, we have repurposed Buccaneer to work with EM data.…”

A global Ramachandran score identifies protein structures with unlikely stereochemistry

“…All software for refining macromolecular models uses a standard set of stereochemical restraints on covalent geometry (Engh & Huber, 2012): these provide sufficient information for structures at 3.0Å resolution or better. Advances in electron cryo-microscopy (Li et al ., 2013; Bai et al ., 2015) have led to greatly improved resolution of cryo-EM maps, but while this improved resolution enabled full-atom refinement of macromolecular structures (Afonine et al ., 2018; Nicholls et al ., 2018), the majority of cryo-EM models are still solved in the 3-5Å resolution range. Likewise, in X-ray crystallography, low-resolution data sets remain an issue: atomic modeling and refinement against low-resolution data is challenging and can benefit substantially from using all available a priori knowledge about the molecule at hand (Kleywegt & Jones, 1998).…”

“…While this metric has been available since 1997 in the WHAT_CHECK program and has been routinely reported by PDB-REDO (Joosten et al ., 2009, 2014), it never made it into mainstream validation pipelines (Read et al ., 2011) nor did it become standard practice to report the metric in the model quality summary in “Table 1”. There is now an avalanche of lower resolution structures being deposited, in a large part thanks to the cryo-EM revolution (Li et al ., 2013; Bai et al ., 2015), and to dramatic improvements in refinement methods (Afonine et al ., 2018; Nicholls et al ., 2018) that now actively exploit the majority of available a priori information about model geometry. The explicit use of the (φ, Ψ) angles distribution in refinement now makes it essential for new model quality measures that are independent of the information used in the refinement target function.…”