MBAR is a free energy estimation method that minimizes the free energy variance and considers the overlap between a given state and all the others in the transformation path , which means that high uncertainties (±3k B T ) suggest the presence of problems in the transformation's path. TI's uncertainty estimates are much lower, but we believe this is an artifact. Error analysis for TI simply does not work the same way and does not give insight into whether exploration of phase space is adequate, unlike MBAR. Specifically, uncertainty estimates from TI usually factor in only the uncertainty in the integrand at each sampled lambda value and could potentially also factor in the smoothness of the integrand (i.e. numerical integration error) but do nothing to factor in whether the integrand will in fact vary smoothly in between lambda points; usually no data is available on this. BAR and MBAR, in contrast, factor in information about how well the intermediate states overlap in phase space and reflect high uncertainties when phase space overlap is poor. In our experience, usually TI would suffer from similar problems if additional intermediate states were added, but uncertainties in TI typically do not reflect this, as is the case here. Thus, the high uncertainty of the MBAR value indicates a sampling/convergence problem which warrants further exploration.