Additionally, we tried using the protocol optimized with ligand 1 to estimate the population of the binding modes for ligand 2. We found that same protocol did not work for ligand 2 (Figure ). Thus, at least for these binding mode sampling problems, umbrella sampling is not only expensive, it cannot be used as a reliable option for estimating binding modes, especially when doing a prospective or blind study, as the protocol employed would require tuning for each individual ligand to ensure correct populations are obtained -a procedure which is impossible when correct populations are not known.