Abstract
A wide range of cellular processes requires the formation of multimeric protein complexes. The rise of cryo-electron microscopy (cryo-EM) has enabled the structural characterization of these protein assemblies. The density maps produced can, however, still suffer from limited resolution, impeding the process of resolving structures at atomic resolution. In order to solve this issue, monomers can be fitted into low- to medium-resolution maps. Unfortunately, the models produced frequently contain atomic clashes at the protein-protein interfaces (PPIs), as intermolecular interactions are typically not considered during monomer fitting. Here, we present a refinement approach based on HADDOCK2.4 to remove intermolecular clashes and optimize PPIs. A dataset of 14 cryo-EM complexes was used to test eight protocols. The best-performing protocol, consisting of a semi-flexible simulated annealing refinement with centroid restraints on the monomers, was able to decrease intermolecular atomic clashes by 98% without significantly deteriorating the quality of the cryo-EM density fit.
Highlights
Crucial processes in our cells such as metabolism, signal transduction, gene replication, and transcription all involve an interplay between proteins
Three common experimental methods to resolve protein structures are X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-electron microscopy, of which cryo-EM is the experimental method of choice to study large functional complexes (Bai et al, 2015; McPherson and Gavira, 2014)
The 14 multimeric cryo-EM complexes included in our dataset (Table 1; Figure 1) were selected from the PDB based on resolution, size and absence of polynucleotides
Summary
Crucial processes in our cells such as metabolism, signal transduction, gene replication, and transcription all involve an interplay between proteins. In order to analyze these multimeric protein complexes and their protein-protein interfaces (PPIs), high-quality structures are required. Three common experimental methods to resolve protein structures are X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-electron microscopy (cryo-EM), of which cryo-EM is the experimental method of choice to study large functional complexes (larger than $80 kDa) (Bai et al, 2015; McPherson and Gavira, 2014). The majority of the maps released originate from single-particle (SPA) cryo-EM (77%), yielding an average resolution of 5.6 A. Other cryo-EM techniques such as tomography, which allows in situ detection, cannot yet reach such high resolutions. As the ex situ conditions of SPA can affect a protein’s conformation, the lower-resolution maps obtained with in situ techniques are valuable for studying proteins under physiological conditions (Schur, 2019)
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