For the decoupling and recoupling of the ligand, we performed both NEQ and EQ calculations. Since these steps are most computationally challenging and expensive, we sought to investigate how NEQ performs compared to EQ. Some of our simulations use a "bubble-ligand" that repels water to overcome water sampling problems during NEQ switching transitions. Below we show why and how it is used. To incorporate this bubble-ligand into the thermodynamic cycle (right part of Figure ), we first have to restrain three buried water molecules and a protein loop close to the binding site. This is necessary because the bubble-ligand creates a vacuum in the empty binding site which some side chains of this loop try to fill resulting in conformational changes of the entire loop (Sec. 5.5.3). After restraining the loop, the bubble-ligand is turned on, so that in the next step, where the ligand is decoupled, water cannot enter the binding site. This separates the water sampling problems from the decoupling of the ligand in HSP90.