Moreover, the nature of such distributions is generally not normal . In this study, we have performed ensemble simulations of large size (whose members contain up to 100 replicas) for a subset of systems using both EQ and NEQ approaches. Figure displays distributions of ∆G com alch obtained from each replica individually for the EQ approach. Figures and) capture the corresponding display of data for the NEQ approach but using a different subset of ligand-protein complexes. It should be noted that the results displayed in Figure were obtained using 50 transitions per replica. Similar distributions and running averages using 10 and 100 transitions per replica are included in the Supporting Information (Figure )). It is evident that the distributions deviate from Gaussian behaviour in : Correlation plots for absolute binding free energies from the non-equilibrium method (600 ps) against those from the equilibrium method for both raw predictions as well as adjusted for ∆G prot conf . 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. many cases. Note that the widths of these distributions vary by over 30 kcal/mol in numerous cases. This in turn implies large uncertainty, especially if one seeks to use ∆G values to select the best ligands in a drug discovery context. Figure displays the effect of transition lengths (1, 2 and 4 ns) on ∆G com distributions; it is evident that transition lengths do not affect the width or nature of these distributions.