(A) Mutational tolerance is highly correlated with species-level sequence conservation and is maximized at EC100 (Spearman's ρ = −0.673, Pearsons r = −0.65; ρ = −0.71, r = −0.69; ρ = −0.74, r = −0.73; ρ = −0.64, r = −0.62; for -Iso, EC50 Iso, EC100 Iso, and EMax Iso, respectively; all p<<0.0001). Here, we calculated sequence conservation using the Jensen-Shannon divergence from a multiple alignment of 55 ADRB2 orthologs from the OMA database. The blue line is a simple linear regression. (B) Similarly, mutational tolerance of the individual positions is highly correlated with sequence conservation across Class A GPCRs and is also maximized at EC100 (ρ = −0.57, r = −0.57; ρ = −0.62, r = −0.63; ρ = −0.68, r = −0.68; ρ = −0.64, r = −0.65 for -Iso, EC50 Iso, EC100 Iso, and EMax Iso, respectively; all p<<0.0001). The blue line is a simple linear regression. (C) Activity measurements for individual substitutions correlates with predictions from EVMutation, and is maximized at EC100 (ρ = 0.37, r = 0.33; ρ = 0.46, r = 0.41; ρ = 0.52, r = 0.48; ρ = 0.50, r = 0.49; all p<<0.0001). The blue line is a simple linear regression. (D) The location of the 100 most deleterious mutations by activity score at EMax Iso on the β2AR snake plot. Mutations are concentrated in the transmembrane domain. (E) Top: Distribution of the 100 most deleterious mutations by activity score at EMax Iso stratified by domain. Bottom: Location of these mutants on the 3D structure of the β2AR. These positions (colored as in D) tend to face into the core of the β2AR (PDB: 3SN6; Gs in blue).