In order to obtain the binding free energy, this bubble approach has to be included in the thermodynamic cycle (Figure , right). This makes the approach not only computationally demanding, but also difficult to generalize. The protocol had to be improved iteratively, gearing it towards this specific system. We had to optimize the van-der-Waals parameters of the bubble-ligand to sufficiently repel the water. We also had to restrain the loop that moved into the binding site to fill the vacuum created by the bubble (Sec. 4.). For a different system it would be difficult to predict such structural changes a priori. Additionally, water sampling problems, although less severe, were still present when turning the bubble-ligand off (SI Figure ). The approach only separated the water sampling challenges from the decoupling of the ligand, but did not solve the problem directly. These factors suggest this approach is not a good general strategy but our results further support our hypothesis that water sampling caused issues in both EQ and NEQ calculations.