The free energies of binding for MIF-180 to MIF calculated with varied sets of Boresch restraints were fairly self-consistent and in good agreement with experiment. However, removal of the orientational restraints produced estimates which were up to approximately 4 kcal mol -1 more negative, likely because the ligand failed to sample all relevant orientations as the LJ interactions were removed. It was found that the calculations were highly sensitive to the sampling of water in the binding site at intermediate stages of vanishing, and that under-hydration of the binding site during these stages over as few as 3 λ windows could shift the binding free energy by over 3 kcal mol -1 towards more favourable binding.