The ABFE of trapped water for one of our HIV-1 Protease systems (PDB ID: 1HPX) was also calculated by the double decoupling method. Two separate studies reported -3.1 ± 0.6 kcal mol -1 and -3.2 ± 0.4 kcal mol -1 as the ABFE of the trapped water in 1HPX. With our protocol, we calculated an ABFE of -5.67 ± 0.5 kcal mol -1 for this system, which remains in disagreement with the previous studies by more than 2 kcal mol -1 . The reason for this discrepancy might be the difference in the treatment of the trapped water between the reference studies and our study. In the reference studies, the trapped water was restrained in an interacting state with a harmonic constraint potential tuned to correspond to the range of motion of an interacting trapped water in the binding site. The contribution of the harmonic constraint potential was accounted for by an analytical correction term corresponding to the translational degrees of freedom of the trapped water. In our calculations, we have simulated a completely unrestrained state of water (stage 1, Figure ) and have accounted for the harmonic position restraint on the trapped water (edge B, Figure ) by explicitly calculating the free energy of the position restraint. Given this important difference in the trapped water treatment, our thermodynamic cycle potentially uses a more clear and well-defined definition of the trapped water's displacement, which may explain the discrepancy in the ABFEs between ours and the reference studies. For the 1HPX system, the study by Barillari et al. reported an ABFE of -10.0 ± 0.5 kcal mol -1 for the trapped water (-8.0 ± 0.5 kcal mol -1 after taking into account the correct standard concentration of water and neglecting the symmetry correction term, as previously described in this section), which reflects too strong binding of the trapped water compared to what we as well as the double decoupling studies observed. The possible reason for this discrepancy might be the use of the hard-wall potential, to prevent solvent flooding and a binding site collapse, in the study of Barillari et al. The hard-wall potential might estimate the binding affinity of the trapped water in the solvent-exposed binding site of the HIV-1 Protease system to be too potent because it reflects the binding free energy of the trapped water in a preexisting cavity.