In order to clearly identify pre-lasso and pre-tadpole structures, we developed the lasso threading classification algorithm (Figure ) combined with the criterion that the distance between the Gly1N and Glu8Cδ be less than 6 Å. While this algorithm unequivocally filters structures into one category or the other, it is not a differentiable variable essential for enhanced free energy sampling, or even a non-differentiable variable useful for plotting the phase space distribution. Instead, we tested the summation of ring-tail distances, using the alpha carbons of residues 1, 3, 6, and 8 to that of residue 19, combined with the Gly1N-Glu8Cδ distance to characterize conformational phase space (Figure ). The resulting 2D folding free energy landscape of all replicas (-𝑅𝑇 ln 𝜌, where 𝜌 is the relative probability) demonstrates that the summation of distances between macrocyclic residues and the penetrating tail residue can distinguish the pre-lasso (basin A), unfolded (basin B), and pre-tadpole (basin C) ensembles. Focusing on only the filtered structures, where Gly1N-Glu8Cδ ≤ 6 Å, all replicas (Figure ) show reasonable separation between the prelasso and pre-tadpole structures, with some degree of overlap as the ring-tail distance starts to increase. The overlapping pre-tadpoles share some conformational similarities with the pre- lassos, like the β-hairpin preserved (𝛬 < 7) and less consistently a hydrogen bond between the backbone of the residues Val6-Tyr20. The ring-tail distance also presents a better separation than Glu8-Ψ when combined with 𝛬 (Figure ), highlighting the 6-20 hydrogen bond occurs with longer 1-8 distances but a tightly formed β-hairpin. While this combination of variables provides useful discretization of phase space, the overlap in pre-lasso and pre-tadpole distributions indicates these are not yet effective parameters for enhanced sampling simulations aiming to distinguish the probability of forming a lasso versus tadpole structure once the isopeptide bond forms. The identification of such parameters will be a valuable advance for future research. Collectively, the folding landscape (Figure ) captures how the formation of the pre-lasso precursor is entropically disfavored. Both the broad unfolded phase space and pre-tadpole distributions dominate over the pre-lasso ensemble, which occupies a much narrower range within these parameters, reflective of the compact structure of the interlocked lasso motif.