Fig. S5.

Single-molecule PB analysis. This method consists of imaging single molecules and counting the number of observed bleaching steps. However, there is a nonzero probability that any given fluorophore will already be bleached (or otherwise unobserved); thus, less than the highest possible number of fluorescence decreases will be detected. Thus, the resulting observations are drawn from a binomial distribution (45). Moreover, in our case, we also have the presence of both nonfluorescent endogenous GR and unliganded haloGR. To estimate the amount of fluorescent GR inside the cells, we first assumed a complete dimeric population in the WT receptor based on the N&B results. Given the fact that we observed 18.98% of two-PB events, we can estimate the fluorescent population at around ∼43% [(0.43)² = 0.1849]. (A) For the WT, we calculated two independent binomial distributions, one for a complete dimeric population (i.e., the nucleoplasm) and the other for a complete tetrameric population (DNA-bound GR). Because the GR-bound fraction is 3.5% (26), we weighted the contributions of each binomial model. Our observations fit well to the expected model, suggesting the presence of a small population of higher oligomerization states for GR. (B) By using the same estimation of the fraction for fluorescent GR molecules, the case for a fully tetrameric P493R did not fit the experimental data. This result suggests that a mixture of different oligomerization states, rather than a fully tetrameric population, is the most likely scenario for this mutant.