Here, we find that sampling problems primarily occurred in the alchemical intermediate states. In the EQ approach, each state has to be sampled at equilibrium, meaning that the correct equilibrium distribution of the Ile78 dihedral angle has to be sampled at every λ window. This can be especially challenging in intermediate states where energy barriers between the rotamers can be high. In SI Figure we show an example of the correlation between the dH/dλ values in one λ window and the dihedral angle of Ile78. The three replicates did not sample the same rotamer distribution of the side chain indicating that sampling time was not sufficient to reach equilibrium.