PT - JOURNAL ARTICLE AU - Ali Ebrahim AU - Blake T. Riley AU - Desigan Kumaran AU - Babak Andi AU - Martin R. Fuchs AU - Sean McSweeney AU - Daniel A. Keedy TI - The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (M<sup>pro</sup>) AID - 10.1101/2021.05.03.437411 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.05.03.437411 4099 - http://biorxiv.org/content/early/2021/11/07/2021.05.03.437411.short 4100 - http://biorxiv.org/content/early/2021/11/07/2021.05.03.437411.full AB - The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or Mpro, is a promising target for development of novel antiviral therapeutics. Previous X-ray crystal structures of Mpro were obtained at cryogenic temperature or room temperature only. Here we report a series of high-resolution crystal structures of unliganded Mpro across multiple temperatures from cryogenic to physiological, and another at high humidity. We interrogate these datasets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps. Our analysis reveals a temperature-dependent conformational landscape for Mpro, including mobile solvent interleaved between the catalytic dyad, mercurial conformational heterogeneity in a key substrate-binding loop, and a far-reaching intramolecular network bridging the active site and dimer interface. Our results may inspire new strategies for antiviral drug development to counter-punch COVID-19 and combat future coronavirus pandemics.Synopsis X-ray crystallography at variable temperature for SARS-CoV-2 Mpro reveals a complex conformational landscape, including mobile solvent at the catalytic dyad, mercurial conformational heterogeneity in a key substrate-binding loop, and an intramolecular network bridging the active site and dimer interface.Competing Interest StatementThe authors have declared no competing interest.Fo-Foisomorphous difference electron density mapCOVID-19coronavirus disease 2019SARS-CoV-2severe acute respiratory syndrome coronavirus 2MproSARS coronavirus main protease