We next had to develop a tool that could definitively distinguish left-from right-handed pre-lasso conformations (Figure ). We developed a handedness descriptor, 𝜅, that leverages the orientation of the ring encirclement relative to the directionality of the tail: if the Nterminus wraps around the tail in a clockwise manner, it is right-handed and vice versa. By projecting the vectors connecting ring alpha carbons onto the plane normal to the tail (Figure ) and evaluating their cross products with the vector connecting tail piercing alpha carbons 8 20 to 19 using a summation of dot products, which makes 𝜅 positive for right-handed conformations and negative for left. Both tools, the lasso threading classification algorithm and handedness descriptor, are fully described in Methods and available at (Lasso Threading Classification and Handedness Evaluation Descriptor).