Thus, charged alchemical regions can be expected to have larger uncertainties when employing the REST2 protocol. In our dataset of 60 ligand pairs, 5 have charged alchemical regions (all binding to BACE (scaffold) system with a charge of +1). Four of them have larger uncertainties when using REST2 than without it. MUEs for these 5 BACE (scaffold) ligand pairs with and without REST2 are 2.74 ± 0.78 kcal/mol and 0.82 ± 0.21 kcal/mol respectively, manifesting the degradation in accuracy on applying REST2 to this system. It should be noted here that all these 5 ligand pairs have uncertainties ranging between 0.75 kcal/mol and 1.3 kcal/mol, and hence form a subset of the 9 ligand pairs with σ T IES > 0.5 kcal/mol as discussed earlier.