To explore the high uncertainty of our MBAR free energy estimates, we examined the degree of overlap the intermediate states had with each other. Phase space overlap analysis quantifies the probability that any given configuration of an intermediate state can be found in other states. A good rule of thumb for designing a set of free energy calculations spanning between two states is to ensure that the states along the path have significant overlap with their neighbors as shown in Fig. . More overlap improves the quality of the MBAR free energy estimation: Fig. (b) represents a set of restraining simulations where the free energy uncertainty can potentially be accurately estimated using BAR and MBAR; Fig. shows a case where it cannot. In our case we find that the α-methanol simulation using 18 intermediate states does not have adequate overlap (Fig. )-specifically, the states 4 ≤ λ i ≤ 17 do not have overlapping configurations with other states, which explains the 3 k B T uncertainty in our MBAR estimate.