Many other simulation studies have used chignolin to test and evaluate enhanced sampling and other methods. Methods Molecular dynamics simulation. MD simulations of chignolin were performed from both native and extended starting conformations, using GROMACS 2020.3 67 on the Folding@Home distributed computing platform. The native conformation was taken from the NMR-determined solution structure (PDB ID: 1UAO). Extended conformations were constructed using AmberTools. Each structure was solvated using 4000 TIP3P water molecules with 100 mM NaCl counterions in a cubic box of length ∼5 nm. For each force field considered, the system was energy-minimized and equilibrated to 300 K for 100 ps. Production-run simulations were performed in the NPT ensemble using a Parrinello-Rahman barostat with 6 Fifty simulations each were starting from native and extended structures, yielding a total of 100 trajectories of various lengths for each force field. To minimize sampling bias, only trajectories longer than 200 ns were selected for analysis. This resulted in a selection of 34 trajectories (17 from extended and 17 from native starting conformations). Distributions of analyzed trajectories for all force fields (Figure ) have an aggregate dataset size average of 27.5 µs, with standard deviation ±220 ns across force fields.