Fig. 3: Structural determinants of energetic couplings inferred from combinatorial mutagenesis.

a, Relationship between folding coupling energy strength and minimal inter-residue side-chain heavy-atom distance. The mean is shown and error bars indicate 95% confidence intervals from a Monte Carlo simulation approach (n = 10 experiments). Points are coloured by binned inter-residue distances (see legend in panel b). Spearman’s ρ is shown for all couplings (top value), as well as those involving pairs of residues separated by more than five residues in the primary sequence (bottom value). Core residues are indicated as triangles. b, Relationship between folding coupling energy strength and linear sequence (backbone) distance in number of residues. The measure of centre and error bars are as defined in panel a. c, Interaction matrix indicating folding energy coupling strength, as well as pairwise structural contacts in GRB2-SH3 (Protein Data Bank (PDB): 2VWF; minimal side-chain heavy-atom distance < 8 Å, black circles). Grey cells indicate missing values (non-mutated residues) and constitutive single aa substitutions are indicated in the x-axis and y-axis labels. Black diagonal lines demarcate residue pairs that are distal in the primary sequence (backbone distance > 5 residues). Reference secondary structure elements (arrow, beta strand) are shown at the top and right. d, 3D structure of GRB2-SH3 (PDB: 2VWF) indicating the top ten energetic couplings with black dashed lines. Combinatorially mutated residues are shown in orange. e, Bar plot showing ranked features from the linear model to predict folding coupling strength. Bar width indicates coefficient significance (P value from uncorrected two-sided t-test). Blue, positive coefficient; red, negative coefficient; grey, non-significant (P > 0.05). f, Correlation between linear model predicted and observed folding coupling strength for training (top) and test (bottom) data. Pearson’s r is shown. The error bands represent the 95% confidence intervals for the predicted values.