We find that using the RMSD to reference crystal structures to define states shows some agreement with our TICA-based approach results in different state categorizations. For each native system trajectory, we calculate the RMSD of each trajectory frame to one of three reference crystal structures and plot onto our 2D TICA surface, color-coding by RMSD. We observe that generally, frames closer to each crystal structure in TICA space also tend to have lower RMSD to that crystal. However, we also observe that state definitions based on RMSD are quite fuzzy. We find that the majority of trajectory frames with low RMSD to the 4w52 crystal structure fall in the right hand region of the oval-shaped well. However, we also observe a number of frames vertically below the 4w52 crystal structure with relatively high RMSD to the structure (Figure ). Frames with low RMSD to the 4w57 crystal structure generally fall in the bridge region, but we also observe a number of frames in the closed and open regions with relatively low RMSD to the 4w57 structure (Figure ). Lastly, we find that frames with low RMSD to the 4w59 crystal mostly fall in the half-moon shaped well in the left region of the plot. However, we find frames close to the 4w57 structure with relatively low RMSD to the 4w59 crystal. Additionally, some frames in the upper left corner of the half-moon well have just as high of RMSD to the 4w59 structure as frames close to the 4w52 crystal (Figure ). Choice of protein starting structure affects sampling of protein conformational states. We proceed to construct Markov state models (MSMs) for each native system to resolve metastable states based on slow dynamics. As described in the Methods, we project all na- ) On top of the density of the concatenated trajectory (light purple), we project the frames from simulations of the (A) 4w52-benzene (purple dots), (B) 4w57-butylbenzene (cyan dots) and (C) 4w59-hexylbenzene (green dots) systems in 2D TICA space. (D-F) For each frame of the concatenated trajectory, we calculate the RMSD of the F-helix to different crystal structures. Shown is RMSD to (D) the benzene-bound crystal structure (PDB ID: 4w52), (E) the butylbenzene-bound crystal structure (PDB ID: 4w57) and (F) the hexylbenzene-bound crystal structure (PDB ID: 4w59). The heatmap signifies the RMSD of each frame of the concatenated trajectory to the specified reference crystal structure. We additionally map where the 4w52 (purple star), 4w57 (cyan star) and 4w59 (green star) crystal structures fall on this landscape for reference. Frames closer to each respective crystal structure in TICA space tend to have lower RMSD to that structure.