Within the TIES protocol, we recommend flexibility in the ensemble size to be used depending on the level of precision desired and the system being investigated. Precision of results increases with ensemble size. However, the exact trend is system-specific, and so is the magnitude of uncertainty associated with the predicted ∆∆G values. Therefore, a fixed ensemble size yields different error bars for different systems. This is evident in our current dataset where 69 ligand pairs have uncertainties greater than 0.5 kcal/mol using ensemble size 5 while 81 are below 0.1 kcal/mol using the same ensemble size. We have already seen in the previous sections that increasing the ensemble size reduces uncertainty in predictions. This is why our protocol leaves the ensemble size parameter adjustable. However, this does not mean that the ensemble size can be arbitrarily small as that affects the accuracy of results. For instance, there are 70 ligand pairs in the current dataset where σ T IES is larger for ensemble size 5 compared to ensemble size 3. Similarly, on closely observing Figure (b), we can see prominent kinks for a few ligand pairs for ensemble sizes below 5. These observations indicate that, for a substantial number of systems, an additional replica beyond the second or third replica may represent a slightly different region of the phase space, and hence increase associated uncertainty. Thus, using an ensemble size of only 2 or 3 does not capture the full picture, leading to over-confident predictions. In addition, as we have seen, the distribution of free energies deviates from Gaussian behaviour; 2 or 3 replicas are simply insufficient to encompass contributions from heavy tails. One is unlikely to get the correct expectation value either, which is the quantity that determines accuracy.