We found that insufficient sampling of different rotamers around the bond highlighted in yellow (Figure ) caused these sampling problems. Rotation around a rotatable bond in compound 03 (Figure ) was slow in some λ windows and depended on whether this ligand started as a dummy or fully interacting. Analyzing the free energy change caused by modifying the vdW/restraints versus the Coulomb interactions separately showed that the difference in the ∆∆G depending on the direction mostly happened in the λ windows modifying the Coulomb interactions. As described in Section 4.2, replica exchange swaps were carried out between all λ windows in legs B and C in the thermodynamic cycle (Figure ) while leg D was run in a separate calculation. In the transformation going from compound 03 to compound 01, compound 03 started as a fully interacting ligand, and only a single rotamer of the R-group (Figure ) was sampled in all λ windows of leg B and leg C. Meanwhile, when going in the opposite direction, from compound 01 to compound 03, compound 03 started as a dummy ligand in leg B, sampling multiple rotamers. Here, in contrast to the transformation in the other direction, two different rotamers were sampled in the λ windows in which the Coulomb interactions were modified (leg C), benefiting from broad rotamer sampling in non-interacting and weakly interacting states through replica exchange. In this example, the rotamer distribution differed across different states in the alchemical pathway, and since for some states the equilibrium rotamer distribution was not sampled, results were incorrect.