In this study, we resolve the discrete states of the T4 L99A binding pocket observed in ex-Figure . MSM-resolved states for each mixed system. We initialize benzene, butylbenzene and hexylbenzene ligands each in their respective non-native crystal structures to observe potential changes in the F-helix. On top of the density of the concatenated trajectory (gray), we project the frames corresponding to simulations of the (A) 4w57-benzene, (B) 4w59-benzene, (C) 4w52-butylbenzene, (D) 4w59-butylbenzene, (E) 4w52-hexylbenzene and (F) 4w57-hexylbenzene systems. We color frames based on their MSM-assigned macrostates, keeping macrostate definitions consistent across the different native systems. On these timescales, simulations of benzene in T4 L99A primarily stay in the closed conformation when simulated beginning from either the (A) 4w57 or (B) 4w59 structures. periment 10 in MD. By selecting a combination of features which capture structural elements of the F-helix, we design a trajectory where the slowest motion of the system corresponds to the opening of the T4 L99A binding pocket. In doing so, we are able map a set of continuous trajectories onto a two-dimensional TICA surface. We find based on the relative positions of the crystal structures in TICA space that the closed and intermediate structures are much closer in distance to each other than to the open state, suggesting greater structural similarity between those two states to each other than to the open conformation. Evidence of the intermediate observed in MD. We find evidence that the intermediate state is a kinetically distinct state in our MD simulations of the 4w59-butylbenzene system. When evaluating the native 4w57-butylbenzene system, we find that the trajectory samples the bridge region of the TICA surface which we believe corresponds to the intermediate state (Figure ). However, this bridge region is not identified as a discrete state by the MSM built from simulations of the native 4w57-butylbenzene system (Figure ) but is by the MSM built from simulations of the 4w59-butylbenzene system (Figure ). This suggests that while the intermediate state may be observed in our MD simulations of the 4w57-butylbenzene system, transitions to and from the state are too transient for it to be considered a kinetically distinct state. However, the region corresponding to the intermediate state is identified as a kinetically distinct state when we attempt to force the transition in simulations of the 4w59butylbenzene mixed system.