We take a subset of 8 different ligand-protein complexes to study the effect of this parameter. Figure : Comparison between ABFE predictions with 10 and 100 NEQ transitions per end-point replica (denoted as "trns/rep") using 10 replicas and 100 replicas at each end-point. The solid red line denotes the perfect correlation, whereas the blue, orange and red shaded regions represent ±1, ± 1-2 and ± 2-3 kcal/mol ranges. tions using 100 versus 1000 transitions in each direction whereas the right panel compares those using 1000 versus 10000 transitions in each direction. It is apparent that the two results agree in some cases whereas differ in others. Thus, 100 transitions is the minimum requirement, but it is insufficient in the majority of cases. For instance, in Figure only 3/4 cases (out of total 8) fall in the blue shaded region for ensemble size 10/100. But which is the optimal way to increase the number of transitions when required: increasing the ensemble size, the number of transitions per end-point replica or some combination of both. We have obtained ABFEs with 1000 transitions in each direction using two different ensemble sizes: 10 (with 100 trns/rep) and 100 (with 10 trns/rep). Figure compares results using both these settings with each other. The corresponding mean signed and unsigned errors are 0.70(0.48) and 1.04(0.39) kcal/mol respectively indicating that the values do not agree too well, although the larger ensemble has smaller error bars and thus better precision. Next, we compare both settings of overall 1000 transitions with that of 10000 transitions (100 replicas × 100 transitions per replica, assumed to be the most reliable) in Comparison between ABFE predictions with overall 1000 and 10000 NEQ transitions (denoted as "trns") in each direction using 10 replicas (100 trns per replica) and 100 replicas (10 trns per replica). The solid red line denotes the perfect correlation, whereas the blue, orange and red shaded regions represent ±1, ± 1-2 and ± 2-3 kcal/mol ranges. each direction in Figure . It is clear that, with smaller MUE, increasing the number of transitions per replica is a preferred way of increasing the overall number of transitions given that a minimum ensemble size is used. This is consistent with our previous observations (Figures and) that enhancing trns/rep brings about the highest reduction in distances. Given that 10 trns/rep is found insufficient in the majority of cases, we recommend using 50 trns/rep as it provides results closer to 100 trns/rep in most cases with half the computational cost (see Figure ). In combination with ensemble size 10 and replica length 10 ns, this leads to overall 500 transitions (chosen uniformly at an interval of 200 ps) for ABFE simulations in the protein environment; whereas for those in the water environment, an ensemble size of 5 should be used leading to overall 250 transitions. As for all other parameters, this number should be flexible and may need to be increased where necessary.