To apply and remove the restraints in the thermodynamic cycles, we performed equilibrium free energy calculations. 3.1. ABFEs of trapped waters. 3.1.1. Our protocol resulted in precise estimates of ABFEs of trapped waters in protein-ligand complexes. We calculated ABFEs of trapped waters in 13 protein/proteinligand complexes (Table ) using the ABFE thermodynamic cycle (Figure ). These 13 complexes are previously studied by Ge et al., and were originally introduced by Barillari et We simulated three replicates of the ABFE thermodynamic cycle for each of the 13 systems, and our calculations resulted in precise estimates of the trapped water ABFEs for all the systems. For each system, our equilibrium free energy calculation to restrain the system resulted in sufficient overlap among the sampled phase spaces of the different states (Figure ). After decoupling the trapped water, we calculated the free energy to remove the restraints, using a second equilibrium free energy calculation. Here, we calculated the free energy to remove the binding site restraints using 7 intermediate λ states between the end states with and without the binding site restraints (see Section 3.1.3 for details). For each system, we obtained sufficient overlap among the sampled phase spaces in the simulations of the intermediate λ states bridging the path between the states with and without the binding site restraints (Figure ). We also obtained sufficient overlap in our NES work distributions for decoupling and coupling the trapped water in all of our calculations.