NTL9, a 39-residue protein with a native structure containing an alpha helix and a beta sheet formed by three beta strands , has a folding process that involves multiple pathways and offpathway conformational states , as supported by the results of AMUSET-TICA. As shown in Figure (a), when employing the same procedure as for alanine dipeptide (i.e., selecting two Gaussian basis functions for each of the top three tICs to construct a rank-343 AMUSET-TICA model, see the Methods section for details), the AMUSET-TICA model accurately predicts the three slowest ITS at lag times as short as 100 ns, which are in agreement with those obtained from a reference 1000-state MSM (referred as the "ground truth" model). The 3 CVs generated by AMUSET-TICA also provide valuable insights into the folding mechanism of NTL9. Figure (b) clearly reveals three highly populated regions on the first two CVs: the native structure (b1), the unfolded structure (b4), and a misfolded structure (b5). The third CV uncovers two additional regions (b2 and b3) along the folding pathway between the unfolded and native states. These five regions correspond to distinct stages in the folding mechanism of NTL9. The primary folding pathway, b4 → (b2, b3) → b1, is consistent with major folding pathways described in previous studies 2, . Figure (b) also identifies several regions outside the main folding pathway (e.g., the b5 region), which is consistent with multiple off-pathway conformational states reported in an earlier study .