Abstract
An evaluation of systematic differences in local structure and conformation in the interior of protein tertiary structures determined by crystallography and by cryo-electron microscopy (cryo-EM) is reported. The expectation is that any consistent differences between the derived atomic models could provide insights into variations in side-chain packing that result from differences in specimens prepared for analysis between these two methods. By computing an atomic packing score, which provides a quantitative measure of clustering of side-chain atoms in the core of the tertiary structures, it is found that, in general, for structures determined by cryo-EM, side chains are more dispersed than in structures determined by X-ray crystallography over a similar resolution range. This trend is also observed in the packing comparison at subunit interfaces. Similar trends were observed in the packing comparison at the core of tertiary structures of the same proteins determined by both X-ray and cryo-EM methods. It is proposed here that the reduced dispersion of side chains in protein crystals could be due to some level of dehydration in 3D crystals prepared for X-ray crystallography and also because the higher rate of freezing of protein samples for cryo-EM may enable preservation of a more native conformation.
Highlights
The 3D structures of proteins play a major role in understanding their functional, regulatory and mechanistic features
We calculated atomic packing scores for every atom buried in the tertiary structures of cryo-electron microscopy (cryo-EM) and crystal entries in our datasets and obtained an average atomic packing score for each structure, after filtering out residues whose packing is affected by missing atoms in the neighborhood and residues with side-chain rotamer outliers
We conclude that the inherent difference in the methods used for sample preparation in cryo-EM and X-ray crystallographic methods likely contributes to differences in packing scores between structures determined using these two methods
Summary
The 3D structures of proteins play a major role in understanding their functional, regulatory and mechanistic features. Over the past few years, cryo-electron microscopy (cryo-EM) has advanced as yet another successful experimental method to determine 3D structures, especially of large macromolecular assemblies (Egelman, 2016; Kuhlbrandt, 2014). Recent advances in technology and methodology involved in single particle cryo-EM (Li et al, 2013; Faruqi et al, 2003) have enabled structure determination of smaller proteins and large assemblies at near-atomic resolution (Merk et al, 2016, 2020; Wu et al, 2020; Hamaguchi et al, 2019). The end goal for protein X-ray crystallography, NMR and cryo-EM is to determine the 3D structures of proteins or assemblies of proteins, the methods themselves are quite different from one another, starting from the sample preparation stage to the structure refinement stage. In the sample preparation stage for NMR, the protein structure is determined in aqueous solution at a temperature above 0C; in cryo-EM, the protein sample in
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