However, in the case of positive errors, the energy barrier between the two holo conformations (such as those corresponding to two different binding modes and/or water occupancy states) may be high such that a simple relaxation may only be able to partially sample one or a few high energy intermediate states leading to a magnification of errors. This issue related to the discrepancy in holo conformations is not specific to the alchemical ABFE methods, but is a general issue that occurs with any MD based method (free energy or otherwise). All such methods are sensitive to the starting structure used and their predictions heavily depend on it. Further, the apparent fact that EQ intensifies the magnitude of (positive) errors does not imply that NEQ is a better approach. It simply means that the NEQ approach is incapable of overcoming the energy barrier to sample other possible conformations, resulting in biased predictions. On the other hand, the EQ approach, by design, enables the crossing of such energy barries and may reduce or even eliminate the bias in predictions given sufficient sampling time. The uncertainty in ∆G exp is also partially responsible for these errors. This means that there might be scope for improvement in these correlations had we access to more comprehensive data on the distribution of