We also calculated the RBFEs for the five ligand pairs with a larger thermodynamic cycle (Table ), shown in Figure , where the re-straints application and removal processes were divided across several different stages, same as in our ABFE thermodynamic cycle (Figure ). Using the larger thermodynamic cycle, we calculated RBFEs within 0.7 kcal mol -1 of the values we obtained from the shorter thermodynamic cycle (Table ). Therefore, we conclude that the simultaneous application of the restraints, and their simultaneous removal, in our shorter RBFE thermodynamic cycle did not result in any significant loss in the accuracy of our RBFE calculations, compared to the incremental application, and removal, of the restraints across several different stages in the larger thermodynamic cycle.