To characterize a lasso folding landscape we first had to develop methods that clearly distinguish regions of phase space that form the threaded isomer (pre-lasso structures) from regions that form the non-threaded variant (pre-tadpole conformations). As discussed below, this has been particularly challenging in past analyses. To address this challenge, we first defined structures capable of forming an isopeptide bond (pre-ring structures) as those with a separation of 6 Å or less between Gly1N and Glu8Cδ. All structures with separations larger than 6 Å were designated as unfolded. We next needed an approach capable of identifying structures in which the tail pierces through the pre-ring. Inspired by the minimal surface analysis used to locate complex lasso proteins from the Protein Data Bank (PDB), we developed a triangulation-based algorithm to evaluate the position of the tail with respect to the pre-ring (Supporting Information -Methods). This analysis divides the pre-ring (residues 1 to 8 of MccJ25) into a series of triangles (Figure ) that are then evaluated for points of intersection by the line segments connecting consecutive residues in the loop/tail (residues 14 to 20) using barycentric coordinates. Although it is not a differentiable variable, this algorithm unambiguously delineates pre-lasso from pre-tadpole conformations.