Our results did demonstrate cases of sampling limitations. This was either seen through poor inter-replica convergence (as shown in some of the MCL-1 ligands), or large deviations from experimental affinities (as shown in HSP90). We note that the ABFE protocol used here is reasonably simple and could easily be improved to address some of these sampling limitations 107 . Indeed, recent work by Khalak et al. demonstrates that non-equilibrium absolute binding free energy can be used to bridge differences between the apo and holo states of a protein, and possibly could alleviate some of the issues shown here.