Here, populations were estimated directly from the PMF and compared to the populations as sampled by BLUES (Figure ), and agreed within statistical uncertainty. This serves to validate that BLUES with sidechain rotations is indeed sampling the correct rotamer distribution and is implemented correctly. ). Error bars were generated using estimates of the standard error from MBAR analysis as provided by the pymbar package. (b) Here the populations of each valine rotamer state from umbrella sampling are plotted in green and populations from BLUES are plotted in blue. Umbrella sampling populations were estimated from the PMF using the Boltzmann relationship (Equation ) Uncertainties were estimated by splitting the umbrella data into 10 chunks and splitting the BLUES data into 5 chunks prior to analysis, and computing the standard deviation in the population estimate across chunks.