FIGURE 4.

pSUFER limitations in designed binders. (a,b) Binders generated through modular assembly and design by grafting an interfacial loop (top) from an unrelated protein and designing the two binding partners. ²⁴ Positions were flagged if mutational scanning suggested at least six amino acid identities with ΔΔG < 0 at the position. Wheat backbone marks missing density on the design model. (a) The interfacial loop failed to exhibit electron density. pSUFER flags Gly26 in the designed loop. (b) pSUFER analysis of the accurately designed des3 flags exposed residues and two interfacial positions. Asn27 is one of the interface loop positions that forms polar contacts with the binding protein and thus may be crucial for function (inset). pSUFER, protein Strain, Unsatisfactoriness, and Frustration findER