The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (M).

The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or M, is a promising target for development of novel antiviral therapeutics. Previous X-ray crystal structures of M were obtained at cryogenic temperature or room temperature only. Here we report a series of high-resolution crystal structures of unliganded M 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 M, 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.