Overall, it seems likely that the use of enhanced sampling methods like HREX impacts sampling in a complex way and will affect equilibrium and nonequlibrium free energy calculations differently. While it seems certain that HREX will help dramatically in some cases, in our tests here it does not provide dramatic ben-efits in terms of overall sampling quality and accuracy. 5.6. Summary of sampling problems in the HSP90 system. In HSP90, water sampling in a solvent exposed binding site caused major problems in alchemical FEC. For the EQ approach, sampling of the correct number of waters in alchemical intermediate states was most challenging while in the NEQ calculations water molecules got trapped in the binding site when quickly coupling the ligand. We attempted to work around the water sampling problem by introducing a bubble-ligand that keeps the water from coming into the binding site. This approach worked to some extent and helped us understand the impact of the water sampling problem better, but is not a good general protocol and more robust methods are required to solve this issue. For both methods, it was crucial to start calculations from protein structures that had three buried binding site water molecules present. Besides the inadequate water sampling, ligand binding mode flips and slow side chain rearrangement caused sampling problems as well. These severe sampling problems highlight the limitation of both approaches in ABFE calculations and reveal clear directions for further investigation.