In one of the three replicates of the simula- tion of the restrained stage of the system (stage 5, Figure ) with the ligand S8, the translation of the ligand was unidirectional (Figure ). We measured this translation by calculating the distance of the nitrogen atom of the p-methylpyridine ring of ligand S8 from the restraint site of the trapped water. In the translated pose, this distance increased to more than 5 Å (as compared to 2.8 Å in the equilibrated starting structure of the simulation). Because of the ligand translation, we did not find any overlap in the NES work distributions of the ligand transformation (edge K, Figure ) that started from the translated pose of the ligand. Because of the lack of overlap, the free energy calculation in that particular replicate was not reliable, and hence, we omitted that particular replicate (i.e., all the free energy values we obtained for the different edges in the simulation of that replicate) from the RBFE estimation and error calculation (see Section 2.6.5 for details) for the BTK system. Additionally, the binding site in the remaining two replicates we considered for the RBFE estimation exhibited the largest fluctuations among any of our RBFE systems. This resulted in an error of 0.5 kcal mol -1 for removing the restraints after the trapped water was decoupled and the ligand water transformed into the bigger ligand (ligand S11, Figure ), ultimately resulting in an overall error of 0.7 kcal mol -1 in the RBFE estimate for the BTK system. The previous study of Wahl et al. has also reported the large fluctuations in the binding site in their simulations of the BTK system.