%0 Journal Article %A Ali Ebrahim %A Blake T. Riley %A Desigan Kumaran %A Babak Andi %A Martin R. Fuchs %A Sean McSweeney %A Daniel A. Keedy %T The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (Mpro) %D 2021 %R 10.1101/2021.05.03.437411 %J bioRxiv %P 2021.05.03.437411 %X 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 a mobile water 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.Competing Interest StatementThe authors have declared no competing interest. %U https://www.biorxiv.org/content/biorxiv/early/2021/05/03/2021.05.03.437411.full.pdf