Complex temperature dependence of residues 192–198 in the P5 binding pocket. (a) Mpro monomer from a cryogenic structure, colored by domain: domain I, residues 8–101, pale green; domain II, residues 102–184, pale blue; domain III, residues 201–303, pale orange. Catalytic dyad residues Cys145 and His41 are shown as sticks (red). Terminal residues are shown in dark gray. P5 binding-pocket linker loop (residues 190–200) shown in dark gray and as sticks (black box). (b) Our new multitemperature structures all have a single backbone conformation for this linker loop region. Regardless of temperature, they all match a similar backbone conformation to the room-temperature 6wqf model (yellow), and not the cryogenic 6y2e model (gray) (Kneller, Phillips, O'Neill, Jedrzejczak et al., 2020) (298* K = 298 K, 99.5% relative humidity). (c)–(e) Phenix ensemble refinement models based on our multitemperature data sets reveal a complex pattern of flexibility that was `hidden' in part (b). (c) For some conditions (100 K, blue; 240 K, cyan; 310 K, red), the ensemble models generally match 6wqf, albeit with variability around the average conformation. For the Ala194–Gly195 peptide (pink arrow), these ensembles match 6wqf. For the Asp197–Thr198 peptide (black arrow), they match 6wqf. (d) For other conditions (277 K, green; 298 K, orange; 298* K, magenta), the ensemble models exhibit shifts away from 6wqf and toward 6y2e. For the Ala194–Gly195 peptide, these ensembles match 6y2e (pink arrow) instead of 6wqf. For the Asp197–Thr198 peptide, they adopt a swath of orientations (black curved arrow) bridging 6wqf and 6y2e. (e) A ∼50° counterclockwise-rotated view of all multitemperature ensemble models, shown as Cα atoms only, illustrates the conformational clustering of the 100–240 K ensembles around residues 193–194 in the P5 binding pocket (bold text), while the 277–310 K ensembles occupy a broader swath of positions within this region.