The overall structure of SARS-CoV-2 main protease at multiple temperatures. (a) A new X-ray crystal structure of apo Mpro at physiological temperature (310 K) (red). The biological dimer involving the other monomer (light-gray surface) is constituted via crystal symmetry. The competitive inhibitor N3 from a previous structure (PDB entry 6lu7) (semitransparent, dark-gray surface) is shown in both protomers for context. (b) Close-up view of the Mpro active-site region, including the catalytic dyad of Cys145 and His41 (red sticks), and highlighting residues that form the substrate-binding pocket (yellow surface). (c) Cartoon putty representation of conformational variability between new Mpro structures described in this work: 100, 240, 277, 298, 298 (99.5% RH), and 310 K. Thickness and color indicate r.m.s. fluctuations (RMSFs) of the Cα-atom positions, from low (thin, dark blue) to high (thick, yellow). The largest differences between the backbones of these structures occur between residues 192–198. Same view as part (a). See also Fig. S3. (d) Heatmap of pairwise Cα-atom r.m.s. deviation (RMSD) between the final refined structures, revealing the temperature-dependent clustering (top right versus bottom left). See also Fig. S2.