In our ABFE thermodynamic cycle, we calculated the free energy of restraining the binding site from a potential collapse. We implemented position restraints on the heavy atoms of the binding site to constrain its shape and postprocessed the trajectories of the unrestrained states to calculate the correct potential to restrain the binding site (see Section 2.3.3 for details). Previously, Ge et al. observed the collapse of the binding site for the BPTI system in their simulations. To calculate the free energy of restraining the binding site from collapsing, they implemented position restraints on the binding site using 19 intermediate λ states between the unrestrained and the restrained states of the system, which made their calculation very lengthy and computationally very expensive. Despite a lengthy protocol, the phase space overlap among the intermediate λ states in their calculations remained low, making their free energy estimate unreliable. In our protocol, we captured the binding site collapse correctly by dividing the binding site restraint removal process using 7 intermediate λ states and reducing the strength of the restraints in each subsequent intermediate λ state. Because our binding site position restraints are origin-dependent (see Section 2.3.3 for details), we post-processed the trajectories of the unrestrained states to calculate the correct free energy contribution for the removal of the position restraints. For the BPTI system, as well as for all the other systems in our study, the post-processing of the trajectories resulted in sufficient phase space overlap among the intermediate λ states (Figures S3 and S5), which in turn resulted in reliable free energy estimates to remove the position restraints on the binding site. 3.2. RBFEs of ligand transformations involving trapped waters. In the present separation of states approach (Figure ), we are interested in deconvoluting the sampling challenges of trapped waters from the calculations of the free energies of ligand transformations. We are interested in calculating the RBFE between two ligands, one of which binds to the protein with a trapped water while the other ligand displaces it. For this, first, using our ABFE thermodynamic cycle (Figure ), we created and stabilized a dry cavity in the binding site of the protein. Then, we revised our thermodynamic cycle to implement a ligand transformation step, in which a bigger ligand is grown in the cavity (Figure ). In the following sections, we present the RBFE calculations for five such ligand transformations.