Encouraged by these results, we then turned to the more challenging FKBP12 enzyme (a cis-trans peptidyl-prolyl isomerase) in complex with an FK506-related ligand. Because the interaction in this system is more complex, we first checked that the ML/MM MD trajectory was physically sound by analyzing 13 key protein-ligand contacts identified in earlier studies. Hydrophobic contacts were defined for three regions of the ligand: the pipecolyl ring, the phenyl ring, and the tertiary pentyl fragment (Figure , panel C). A contact was considered present if the distance between these regions and the side chain of the corresponding amino acid was 4 Å or less. For hydrogen bonds, a distance of 3 Å or less between the acceptor and the donor hydrogen was used as a cutoff. Specifically, uncorrelated frames from both the ML/MM MD simulation and an equally long MM/MM trajectory were analyzed to identify hydrophobic contacts between the tertiary pentyl fragment and Ile56, Leu97, Tyr82, and Ile90; between the phenyl ring and His87, Ile56, and Tyr82; and between the pipecolyl ring and Phe46, Tyr26, Trp59, and Phe99. Additionally, we identified hydrogen bond interactions with Ile56 and Ile91 backbones, respectively. Hydrogen bonds were observed in nearly all frames, and the number of hydrophobic contacts was similar across both trajectories (Figure ).