(A) Number of beneficial mutations identified in each condition based on selection coefficients more than two standard deviations greater than wild-type synonyms. Beneficial mutants at 37°C and in diamide are listed in Figure 5—source data 1. (B) Distribution of the number of beneficial mutations at the same position in both 37°C (left) and diamide (right) conditions (●) is greater than expected if they had occurred independently (◊). Independent expectations were calculated as the probability of the stated number of mutations occurring at the same position by chance. * Indicates observations that were significantly (p<0.01) greater than independent expectations based on random simulations and one-tailed t-tests. (C) Location of positions with four or more beneficial mutations. Positions that are unique to diamide or 37°C are shown in cyan and two shared positions are shown in magenta. (D) The solvent accessible surface area (SASA) of beneficial mutations at 37°C and in diamide compared to all mutations. Distributions are significantly different as measured by a two-sample KS test (37°C vs. all data, N = 270, 12393, p<0.0001, D = 0.2851; Diamide vs. all data, N = 60,12393, p<0.0001, D = 0.3465) (E) The fraction of Hsp90 positions at interfaces that were beneficial in 37°C and diamide conditions. (F) Selection coefficients in standard conditions for beneficial mutations at 37°C and in diamide compared to wild-type synonyms. (KS test; 37°C vs. WT synonyms: N = 463, 660, p<0.0001, D = 0.3281; Diamide vs. WT synonyms: N = 353, 660, p<0.0001, D = 0.3809).