We obtained precise estimates of the ligand RBFEs. We calculated RBFEs (Table ) for five ligand transformations with our RBFE thermodynamic cycle (Figure ). In our calculations, we simultaneously applied the harmonic restraint on the trapped water, the solvent repulsion term, and the position restraints on the protein's binding site (edge M), and calculated the free energy of applying these restraints using an equilibrium free energy calculation (see Section 2.6.3 for details). The equilibrium free energy calculation consisted of two states of the system: an unrestrained and a restrained state (stages 1 and 4, respectively). From the restrained state of the system, we decoupled the trapped water and transformed the ligand in two subsequent NES transformations (edges E and K, respectively). All the restraints, except the positional restraint on the trapped water, were removed simultaneously (edge L), and we calculated the free energy of removing the restraints using another equilibrium free energy calculation comprising of a restrained and an unrestrained state (stages 5' and 7', respectively), however with the transformed ligand.