There are two routes to deal with this issue. First, one can increase the ensemble size in order to better control uncertainty and, second, one may relax the standard charge tolerance of 0.1e to reduce the size of the alchemical region so as to exclude the two carboxylate groups. Our results from both these approaches, compared with the standard one, are shown in Figure . As expected, the error bars for both are smaller than those for the standard protocol. σ T IES falls in the range of 0.7-1.7 kcal/mol for ensemble size 20 using the same alchemical region, compared with 0.2-0.8 kcal/mol for the smaller alchemical region. Thus, TIES predictions using a smaller alchemical region (keeping ensemble size as 5) are more precise than those using ensemble size 20 (but the original alchemical region). However, relaxing the charge tolerance criteria (up to 0.14e for both individual atomic charges as well as overall MCS in this case) compromises the accuracy of the predictions. RMSE increases from 1.91 ± 0.45 kcal/mol to 2.29 ± 0.59 kcal/mol when using the smaller alchemical region.