Characterizing the lasso folding landscape requires both extensive sampling and discretization into regions that distinguish relevant states-in this case pre-lasso, pre-tadpole, and unfolded conformations. This is typically done by identifying a set of distinguishing features or combined features, called collective variables. Although the pre-tadpole was not distinguished as a distinct state in the previous work, we first tested the proposed Glu8 Ψdihedral angle combined with the Λ to see if they might differentiate pre-lasso structures (Figure ). The probability distributions show that separating and classifying pre-lasso and pre-tadpole is not possible with these parameters. There is significant overlap in both the Λ values, which are small indicating the presence of the β-hairpin for all pre-lassos and many pre-tadpole structures, and Glu8 Ψ, which shows both conformations cluster around -40° and 150° (Figure ). Thus, new descriptors were needed to distinguish pre-tadpole from pre-lasso conformations.