SepTop with a standard RBFE method. We compared SepTop with standard RBFE using the non-equilibrium switching (NES) method as implemented in Orion 64 (Orion floe "Equilibration and Non Equilibrium Switching") using the default 6 ns equilibration of the end states and 80 nonequilibrium switching transitions with 50 ps switching time. We used the same ligand map and force field parameters as with the SepTop protocol. We only included ligands with measured binding affinities within the assay limit in our analysis as we did above. We found that SepTop produced better correlation and error statistics than the more standard NES approach (Figure ). SepTop used more sampling time than NES Orion (for 30 transformations: Bound state: 6000 ns vs. 336 ns; Unbound state: 2240 ns vs. 336 ns) which, in addition to problems detailed below, might be part of the reason why SepTop performed better on this system. Since it would have been cost prohibitive to increase the length of the simulations on Orion to the simulation time used in the SepTop protocol, we decided not to perform a direct comparison at equal sampling time. The ∆∆G values calculated using the two different methods mostly agree well with one another, as can be seen in SI Figure and SI Figure . The outliers in the plot indicate that for some of the transformations the two methods did not agree with each other. Two of those outliers involved transformations going from a hydrogen (Pfizer-01-05) or a methyl group (Pfizer-01-04) to a cyclopropyl (Pfizer-01-07). The overlap of forward and reverse work distributions of the nonequilibrium switching transitions was poor (SI Figure ), indicating insufficient sampling of important motions. A third outlier was a transformation that involved the inversion of a chiral center which was potentially not treated correctly in the NES protocol, as discussed below. For the two outlier transformations Pfizer-01 → compound 02 and Pfizer-01 → compound 03, NES predicted a more dramatic change in potency than Sep-Top. These ligands contained the R-group that had caused sampling problems due to slow rotamer sampling in SepTop (see Section 5.3.1) which potentially also caused problems in the NES approach.