Similar to the T4 lysozyme systems, we checked the impact of the Boresch-style restraints on the dissipation by running calculations with a weaker force constant (1/4 of the original restraint strength). SI Figure shows the comparison of these two protocols. We found that reducing the restraint strength did not have a huge impact on dissipation and the overlap of the work distributions. The average dissipated work W d in the reverse process was 18.7 kcal/mol for the protocol with a weaker force constant and 15.2 kcal/mol for the original protocol and in the forward direction 11.5 vs. 13.9 kcal/mol, respectively. 5.5.3. Addressing inadequate water sampling. The water sampling problem can be separated from the decoupling of the ligand by introducing a bubble-ligand that repels the water and keeps it out of the binding site. This . Running average of dH/dλ values (pink, averaged across 5000 data points) and the RMSD of the HSP90 ligand (green) as a function of the simulation time. We show data for λ 21 (second last state) for three independent replicates as an example, figures of all λ windows can be found in the SI. The correlation between dH/dλ values and the RMSD of the ligand indicates that the binding mode flip had an impact on the ∆G estimate, even though these only occurred when the ligand was weakly interacting.