The uncertainty in the work done by each restraint in each simulation window, σ(λ i ), was estimated using blocking analysis , in a manner which has been shown to yield good agreement with uncertainties obtained from independent replicates . In particular, rather than looking for a plateau in the SEM as the size of the blocks increased, as originally described by Flyvbjerg and Peterssen , we instead use the largest standard error of the mean (SEM) obtained for any block size. This avoids the requirement of detecting a plateau and yields a more conservative estimate; i.e., a larger SEM. Then, using Gaussians with the mean and SEM of in each window, new values of were bootstrap sampled for each window 100,000 times and combined to create artificial data for 100,000 notional APR calculations. These were integrated across all windows with splines to generate 100,000 estimates of ΔG°. We report the mean and standard deviation of these 100,000 results as the final mean and its SEM. The SEM of ΔH was computed from the SEM of the total potential energy in each end point window, estimated using blocking analysis, added in quadrature. The standard error of the mean of -TΔS° was calculated using the uncertainties in ΔG° and ΔH added in quadrature.