To ensure we had comparative sampling, despite much longer unbiased simulations, we additionally performed temperature REMD with comparable parameters to those previously used. We were able to obtain 0.82% pre-lasso structures at 300K. Including the filtered structures (those with Gly1 Glu8 distances less than 6 Å) from higher temperatures, the percentage of pre-lassos decreases to 0.08% (Table ), supporting the notion that entropy disfavors the pre-lasso fold. However, all the obtained pre-lassos were right-handed, consistent with those observed in nature. Collectively, these results suggest that the right-handed MccJ25 pre-lasso conformation is a metastable state that is accessible from the unfolded landscape, while the left-handed MccJ25 pre-lasso conformation is inaccessible. Additionally, the pretadpole structures significantly outnumber the pre-lasso structures, consistent with experimental observations of only tadpole structures from de novo folding. These contrasting results to those previously reported led to an investigation of the potential influence of the force fields used. The same system and simulation parameters were implemented with three other modern protein force fields: AMBER ff14SB, 46 CHARMM36, and CHARMM36m. The number of filtered structures where Gly1N to Glu8Cδ is ≤ 6 Å was similar among the different force fields, all with right-handed pre-lasso structures. Relatively more pre-tadpole and unfolded structures were obtained in the simulations with both CHARMM force fields, but this was expected with CHARMM36m since it is optimal for intrinsically disordered proteins (Table ). We also compared contact maps of the first eight residues (Figure ) and found a similar interaction pattern among the tested force fields, with a slight increase in the side chain interactions for the AMBER force fields. Thus, the results between each of the modern force fields tested were consistent. Next, we used the GROMACS 2019.4 simulation engine to repeat the previously reported simulations with the same force field. In these simulations, the right-handed pre-lasso rapidly transitions to unfolded (11 ns) and later to left-handed pre-lasso structures (3.0 µs). These results verify that the force field used in the previous study was the cause of the unexpected findings. GROMOS 43A2 50 was a state-of-the-art force field in 2010 but has since been updated and reparametrized to address numerous issues, especially new Φ/Ψ torsional angle terms. Thus, previous predictions of left-handed folding were due to force field artifacts. MccJ25 folds into the native right-handed pre-lasso structure with modern force fields, suggesting de novo lasso folding can be accessed without the aid of an enzyme, albeit rarely.