Structural origin of differences in the thermostability of WT and F58I, shown by a rigid cluster decomposition of WT (a) and F58I (b) at 316 K.

Rigid clusters are shown as uniformly colored bodies. The mutation site (residue 58), shown by a cyan (a) and a magenta (b) surface, is part of the largest rigid cluster (blue) in both WT and F58I. Hydrophobic contacts in the proximity of the mutation site between carbon atom pairs at most 3.8 Å apart are shown as green (WT) and red (F58I) dashed lines (c). Residues involved in making such contacts are shown as cyan (WT) and magenta (F58I) sticks. Differences in the stability of “rigid contacts” between variant F58I and WT are depicted on the variant structure (d). Two residues form a “rigid contact” if they belong to one rigid cluster. A red (blue) stick connecting C_α atoms of two residues indicates that a rigid contact in the variant is more (less) stable than in the WT (see color scale). Only those contacts of variant F58I that are stabilized or destabilized by ≥ 2 K are shown for clarity; for the same reason, contacts between two residues of the same secondary structure element are not shown. The mutated residue I58 is displayed by magenta sticks. Blow-ups of panel d showing the contact stability between secondary structure pairs mentioned in the main text can be found in S5 Fig.