Previous work on microcin (MccJ25), a bacterial RNA polymerase inhibitor suggested it forms an unnatural left-handed pre-lasso peptide (Figure ). This was unexpected given that MccJ25 and every other structurally characterized lasso peptide is righthanded in nature (Figure ). Herein we revisit the folding propensity of MccJ25 using modern simulation approaches to enable the development of generalized tools for studying pre-lasso folding and supporting future lasso peptide chemical synthesis efforts. We find that native MccJ25 forms the anticipated natural right-handed pre-lasso conformation, and verify that the previous findings were a consequence parameter issues in the GROMOS96 43A2 forcefield. We present new tools that unambiguously distinguish pre-lasso from non-lasso structures and differentiate the right-and left-handed forms. We further describe several collective variables that discretize the pre-lasso folding landscape for distinguishing the threaded pre-lasso from the non-threaded pre-tadpole conformations. We find that the barrier for escape from the pre-lasso conformation is only ~1.5 kcal/mol while the probability for reaching the pre-lasso state is less than 1% for the native sequence. Thus, the probability of de novo folding is extremely low. However, with the addition of chemical modifications to the isopeptide bond-forming residues, we stabilize the pre-lasso fold.