Lastly, we determine there is only a single state sampled in simulations of the 4w59hexylbenzene system (Figure ). While there are motions in the system occurring on slow timescales (Figure ), they correspond to fluctuations within the half-moon shaped well, and are primarily described by differences in the second tIC (Figure ). Given we primarily attribute the F-helix opening motion to the first tIC (Figure ), we do not consider these to be states relevant to our analysis. Simulation of mixed protein-ligand systems allows for estimation of discrete state transition timescales. In attempts to observe transitions within our simulation timescales, we also simulate and build MSMs for a series of mixed systems. These are simulations begun from a crystallographic state belonging with a different ligand than the one they are simulated with, so we expect these simulations to transition to a different state while they are run, even if only once (see Methods for more details). We find that simulations of both the 4w57-benzene (Figure ) and 4w59-benzene (Figure ) systems primarily sample the region of TICA space we believe corresponds to the closed state. For the 4w57-benzene system, we observe a single slow motion (Figure ) corresponding to an exchange between two regions of the oval-shaped well (Figure ).