Challenges encountered here in ABFE will probably effect RBFE as well. Some of these challenges will probably affect relative binding free energy calculations (RBFE) as well. For instance, slow side chain rearrangements will pose challenges whenever different ligands induce slow side chain rotations, as in the case of toluene vs. 3iodotoluene, here, which are not that dissimilar from R-groups that might be modified in RFBE calculations on a larger, drug-like ligand. Water sampling can also cause severe issues in relative binding free energy calculations when a buried water molecule is present with one ligand and displaced by another one. If ligands have a similar shape or displace the same water molecules, RBFE will probably converge faster than the absolute protocol. Binding mode flips, on the other hand, are less likely to occur with RBFE since those happen mostly in the alchemical states where the ligand is only weakly interacting in the binding site. Still, the same issue can affect R-groups of drug-like ligands. 6.2. Strategies to assess sampling problems in alchemical FEC. Sampling problems in these systems helped us to establish some general strategies on how to diagnose and test for problems in alchemical FEC. First, we found that in both EQ and NEQ, running independent replicates is important to identify inadequate sampling and to obtain uncertainty estimates. The analytical error -as well as bootstrapping in NEQ -underestimated the true error.