Implications for future binding studies. One caveat of this study is that the procedure presented may not be suitable for some biological systems. For the T4 L99A system, we have access to crystal structures for all discrete states, allowing us to effectively reverse engineer a trajectory in which the slowest motion of the system corresponds to the opening of the F-helix observed in experiment. In many cases, it may not be known what prompts conformational changes or whether the system undergoes any conformational change at all on binding different ligands. Furthermore, we find in this study that even when running MD for timescales much longer than those run for typical binding studies, our native system trajectories do not sample any protein conformational changes. Thus, simply running longer simulations may not resolve issues with sampling.