Validation of X-ray Crystal Structure Ensemble Representations of SARS-CoV-2 Main Protease by Solution NMR Residual Dipolar Couplings.

Considerable debate has focused on whether sampling of molecular dynamics trajectories restrained by crystallographic data can be used to develop realistic ensemble models for proteins in their natural, solu-tion state. For the SARS-CoV-2 main protease, Mpro, we evaluated agreement between solution residual dipolar couplings (RDCs) and various recently reported multi-conformer and dynamic-ensemble crystallo-graphic models. Although Phenix-derived ensemble models showed only small improvements in crystal-lographic Rfree, substantially improved RDC agreement over fits to a conventionally refined 1.2-& DBLBOND;A X-ray structure was observed, in particular for residues with above average disorder in the ensemble. For a set of six lower resolution (1.55-2.19 & DBLBOND;A) Mpro X-ray ensembles, obtained at temperatures ranging from 100 to 310 K, no significant improvement over conventional two-conformer representations was found. At the residue level, large differences in motions were observed among these ensembles, suggesting high uncertainties in the X-ray derived dynamics. Indeed, combining the six ensembles from the temperature series with the two 1.2-& DBLBOND;A X-ray ensembles into a single 381-member "super ensemble" averaged these uncertainties and substantially improved agreement with RDCs. However, all ensembles showed excur-sions that were too large for the most dynamic fraction of residues. Our results suggest that further improvements to X-ray ensemble refinement are feasible, and that RDCs provide a sensitive benchmark in such endeavors. Remarkably, a weighted ensemble of 350 PDB Mpro X-ray structures provided slightly better cross-validated agreement with RDCs than any individual ensemble refinement, implying that dif-ferences in lattice confinement also limit the fit of RDCs to X-ray coordinates. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).