(A) Top: cartoon of the domain organization of Ir75a; bottom: protein model of DmelIr75a, generated by ab initio protein folding by AlphaFold (Jumper et al., 2021). A magnified view of the ligand-binding domain (LBD) is shown on the right, in which the three functionally important amino acid positions that differ between DmelIr75a and DsecIr75a are highlighted in orange. (B, C) Quantification of responses of the indicated receptor versions expressed in the Ir decoder neuron. (D) Loadings of the first principal component of all responses shown in (B, C). (E) Responses of each of the indicated receptor versions plotted in the C2 vs C4 space. (F) Visualization of epistasis and accessible mutational pathways. Each of the individual, double, and triple mutations are plotted in the first principal component (PC1) axis (error bars are standard error of the mean). Possible evolutionary paths join these mutants with lines. Solid lines indicate when the path joins two points that significantly increase PC1 value (i.e., increased responses to C4 and C5 and decreased responses to C2); dashed lines denote paths that, while accessible, do not lead to significantly increased PC1 values (see Source data, for statistical values). Red lines indicate the cases where two mutations interact epistatically when combined, that is, the combination of the two mutations is not equal to the expected response if their effects added linearly (see Materials and methods for details and source data for statistical values).