In the NEQ approach, water molecules got trapped in the same buried space that caused problems in the EQ approach (Figure ). Particularly, when the ligand appeared in the solvent-filled binding site too quickly, water molecules were not displaced properly and got stuck between the ligand and the protein. This led to poor overlap of the probability distributions of forward and reverse work values and therefore an imprecise (and potentially biased) estimate of the free energy difference. Long transitions were necessary to properly displace the water molecules upon ligand binding. When running each transition for 10 ns, most transitions (63%) correctly ended up with the three buried water molecules in the binding site, for shorter transitions less did so (5 ns/transitions: 41% and 1 ns/transition 7%). But such transitions are computationally expensive and remove much of the benefit of the non-equilibrium approach. 5.5.2. We identified other slow DOF in this system. A second sampling problem in this system was a binding mode flip that occurred when the ligand was decoupled or only weakly interacting (Figure ). In this case, orientational restraints only included atoms from two of the three aromatic rings of the ligand (see spheres, Figure ), hence the third ring was able to flip to a different location.