In addition to the use of non-identical replicate calculations (Section 3.2), we used two different methods, previously detailed in Henriksen, et al. , to estimate uncertainties based on the individual trajectories. Using the approach described by Shirts and Chodera , the statistical inefficiency is determined from the autocorrelation function of the energy to create a subsampled data series that is uncorrelated, at least in principle. The standard error of the mean (SEM, σ) is then computed for the resulting uncorrelated series. Blocking analysis is another approach to estimating the uncertainty of the time series of potential energies, where block-wise SEMs are computed for successively longer blocks of energies. On a plot of SEM vs. block size, a plateau is generally seen for simulations that are considered converged, and the SEM value corresponding to the plateau is taken as the error of the estimation. However, we have observed that, especially when a clear plateau is absent, a more conservative (i.e., larger), and more reliable error estimate is the largest SEM reached for any of the block sizes tested , so we use the latter metric.