Interestingly, the magnitude of errors is consistently lower in case of the EQ approach when their sign is negative, whereas the opposite is true for positive ∆G prot conf values. This makes sense given that the system is allowed to relax at each intermediate alchemical state while staying in equilibrium in the case of the EQ approach, which is not achievable during the short out-of-equilibrium simulations in the case of the NEQ approach. In case of negative errors, two conformations of the protein in its apo state (true/experimental apo structure versus holo structure with a ghost ligand) are connected by a minimal energy barrier and such a relaxation in the EQ approach allows the sampling of experimental apo conformation (the "global minimum"), thereby narrowing the difference between experimental and calculated free energies in the apo state.