Between the BACE1 ligand pair, we calculated an RBFE of 0.32 ± 0.6 kcal mol -1 , which is within 1 kcal mol -1 from the experimental value, though, contrary to the experiment, our calculations estimate the binding free energy of the ligand C4j to be too favorable compared to that of the ligand C4d. We found that our calculations were affected by some sampling challenges that could explain the difference between our RBFE estimate and the experimental value. These sampling challenges are also reported in previous studies of BACE1. 66-68 Keränen et al. 66 studied BACE1 ligands structurally similar to the ligands in our study and reported that the sampling of opening and closing of the 10s loop and the motion of the binding site flap in the BACE1 receptor were crucial to the accuracy of their ligand RBFE calculations. They further showed that their simulations resulted in accurate results when both the open and closed states of the 10s loop were sampled, compared to when only one of the two states was sampled. Another study by Baumann et al. made similar observations, calculating differences in ligand RBFEs up to 3.5 kcal mol -1 depending on whether simulations were started from the open or the closed conformation of the 10s loop of the BACE1 protein. In our BACE1 simulations, we observed that all of our replicates sampled only the closed conformation of the 10s loop, which can be a probable cause for the inaccuracy of our RBFE estimate between the BACE1 ligand pair. The previous studies suggest that to truly converge our free energy calculations, we would need substantially longer simulations of all of the stages of our RBFE thermodynamic cycle to capture these loop motions. 3.2.4. Changes in the binding mode of the BTK ligand resulted in non-overlapping NES work distributions for the ligand transformation . In the simulation of the BTK complex, we observed a translation of ligand S8 (Figure ) in the binding site. Similar to the BACE1 receptor's binding site, the BTK receptor's binding site can also accommodate a rotation of the ligand's p-methyl-pyridine ring, as well as a small translation of the ligand S8. Upon decoupling of the trapped water, the hydrogen bond between the trapped water and the nitrogen atom of the p-methyl-pyridine ring of the ligand S8 vanished. Due to the absence of the hydrogen bond, we observed slight rotations of the p-methyl-pyridine ring and small translation motions of the ligand S8 in the binding site away from the ligand's binding pose in the equilibrated starting structure of the simulations, in all three replicates of the BTK system.