Instead, the negative offset is very likely due to the failure to sample all relevant orientations at intermediate values of λ during vanishing. The close agreement between B1-o and B1-d suggests that the offset is due to the removal of the orientational component of the restraint. As the LJ interactions are removed, the sampling of orientations different to that of the binding pose will become favourable. The gradient of the free energy change as the LJ interactions are removed will likely be less positive in these alternative orientations, because they were high in energy when the LJ interactions were at full strength. Therefore, failure to sample these orientations due to large barriers should give excessively positive free energies of vanishing, resulting in erroneously negative free energies of binding, as is observed.