Backbone structural variability of ensemble models along the Mpro sequence as a function of temperature. (a) RMSF of the backbone Cα-atom positions is plotted versus residue number for each of the different structures in our multitemperature series (colors in legend). RMSF spikes at the N-terminus, C-terminus, and β-turn 153–157 (in contact with the C-terminus) in the ensemble models are truncated in this plot, and should be interpreted with caution. (b)–(e) Backbone structures from ensemble refinement are shown for regions coinciding with temperature-dependent RMSF peaks. The refined single structure is shown as a cartoon, while atoms in the backbone of ensemble models are shown as lines.