Equilibration detection was performed after the adaptive allocation of sampling time was complete. This was done by splitting all time-ordered data for a stage into 100 blocks, so that each contained the same fraction of data (but not necessarily sampling time) from each λ window. The free energy change at each percent of total stage simulation time was computed using the Multistate Bennett Acceptance Ratio (MBAR) as implemented in pymbar 3.1.1. The mean ∆G estimates for the first 10 % and last 50 % of the data were calculated for each run and a paired t-test was performed on the per-run differences. This was repeated after discarding 17 %, 33 %, and 50 % of total simulation time. Equilibration was equated with the first p-value greater than 0.05. Unequilibrated data were discarded and ∆G o Bind was calculated with pymbar. The result was expressed as a mean over all N replicates and uncertainties were calculated as 95 % t-based confidence intervals (using N -1 degrees of freedom). Where not provided, uncertainties in experimental affinity data were assumed to be 0.5 kcal mol -1 . 115,116