Overall, the free energy surface computed via msesMD appears to predict similar stabilities to the profile obtained from 10 s simulations (Figure ); the exception is the G 4 well, where the free energy is predicted as 2 kcal mol -1 less stable than from unbiased MD. We note from the dihedral time series of the 10 s simulations, the lifetime of the G 4 state occupancy is disproportionally longer in the first replicate trajectory (black in Figure ) compared to the other two unbiased trajectories (blue, red). As the G 4 state is sampled only once briefly in each unbiased trajectory, it is not possible to discern if this constitutes an outlier; however it does highlight the fact that the free energy estimates for the G 4 region via unbiased MD are limited due to inadequate sampling.