For the ABFE calculations, we generated 100 starting structures for the NES switches from the last 4 ns of the 6 ns production runs of stages 4 and 5. In each of the NES switches for water decoupling/coupling, the vdw and Coulomb interactions were turned off/on simultaneously over 250 ps by updating the λ value on every time step (∆λ = 0.000008).