Given the chaotic nature of MD , it is necessary to perform ensemble simulations in order to control uncertainties associated with its predictions and ensuring reproducibility of results, thereby making them reliable . Therefore, in this study we employ our TIES approach which involves performing ensemble simulations. The size of the ensemble is a parameter whose value cannot be arbitrarily chosen as is the usual practice among those research groups that deign to perform multiple independent simulations (3 is the most common choice) . In fact, the ABFE thermodynamic cycle in Figure is a workflow so each step needs separate considerations as far as ensemble size is concerned in order to keep the overall uncertainty under control. This means that, for the ligand decoupling step in solvent alone, relatively smaller ensembles could be used without compromisig the accuracy and precision of predictions. Gapsys et al. varied ensemble size from 3 to 5 for the EQ method and 5 to 20