Because of differences in force fields and methods between the reference study and our present work (described below in this section) and the difficulties of validating these methods, we take the literature values only as reference and not as 'ground truth'. The main goal of our ABFE calculations was to develop a robust protocol that ensures good phase space overlap in our equilibrium free energy calculations (see Section 2.2 for details), yields precise and well-converged ABFE estimates, and can readily be expanded to RBFE calculations, rather than to reproduce the reference values from the literature. The study by Barillari et al. presented a good starting point to think about a correct sampling protocol for the decoupling of the trapped water, which is a precursor for a subsequent ligand transformation in our separation of states approach (Figure ).