Average protein-ligand complex ΔG, solvated ligand ΔG, and overall ∆∆𝐺 𝐴→𝐵 𝑏𝑖𝑛𝑑 of the shortrun simulations and long-run simulations with all three equilibration methods are tabulated in from their long-run counterparts than those obtained for Ligands 1-3, with absolute error of 0.6-1.1 kcal/mol compared to 0.1-0.2 kcal/mol, respectively. For Ligand 4, the protein-ligand complex step was the major contributor to the absolute error (0.6-1.0 kcal/mol), while the contribution of solvated ligand step was considerably smaller (0.1-0.3 kcal/mol). This deviation did not alleviate with more simulation time in Protocols A-D (Fig. ), indicating that the relevant timescales are outside the length of these short simulations (see Table for protocol details). The larger error obtained for Ligand 4 can be explained by the difficulty of the mutation, which involves the mutation of the amine group into the methylaminosulfonyl group, as well as a benzene ring methyl group into a chlorine and a naphthalene ring hydrogen into a chlorine. This involves the mutation of seven heavy atoms as well as the overall addition of two rotatable bonds (Fig. ). The methylaminosulfonyl group, which is flexible and solvent exposed, covers a larger area of the phase space and is thus more difficult to converge and requires more simulation time, as seen in To evaluate this discrepancy, we tested Protocol E on Ligand 4 (see Table for protocol details), which yielded mean values of -6.61 ± 0.12, -5.01 ± 0.04, and -1.60 ± 0.12 kcal/mol for the proteinligand complex step, the solvated ligand step, and the overall calculation, respectively. Protocol E As seen in Figure , the most significant accuracy gains were observed during the first nanosecond of production simulation time for Protocols A and B, whereas Protocol C displayed more muted gains. Protocol C displayed elevated inaccuracy at 1 ns of production simulation time when compared to Protocol A and B at the same time period, despite the fact that all protocols are equivalent at this point (see ABFE PLpro. Three 100 ns simulations were performed at each λ-window. The gradients of the short-run simulations were evaluated against those of the long-run simulations in an analogous manner as described for the RBFE simulations. Similarly to the RBFE PLpro study, the amount of simulation time applied to each λ-window varied by λ-window, simulation protocol, and alchemical step (see Fig. ). We note once again that λ-windows in the protein-ligand complex and restraint addition alchemical steps with elevated simulation times in Protocol A tended to have elevated simulation times in Protocols B and C, with an analogous pattern holding for Protocols B and C (see Table for protocol details).