The free energy profile constructed from the triplicate unbiased 10 μs simulations provides a reasonable approximation of the ψ F torsional PMF (Figure ), although the relative stability of the F 2/3 and F 4 wells is predicted as close to degenerate. From the unbiased simulations, the F 1 → F 2/3 transition also appears to have a near negligible barrier height, approximately 2 kcal mol -1 lower than the one predicted via umbrella sampling. The triplicate 1 μs simulations unfortunately fail to adequately sample the complete range of the  F coordinate, but otherwise agree with the 10 μs simulations. Using msesMD simulation, the estimated free energy profile is similar to the unbiased simulations, although the msesMD barrier heights for the F 2/3 → F 4 and F 4 → F 1 transitions are in closer agreement with the PMF from umbrella sampling. Nevertheless, it is noted that msesMD predicts the stability of the F 4 region to be 0.7 kcal mol -1 lower than the umbrella sampling and unbiased MD values. As found in the unbiased simulations, the free energy barrier for F 1 → F 2/3 is predicted by msesMD to be minimal, much lower than found via umbrella sampling. This discrepancy may be due to inadequate sampling of the φ F rotation in the 1-D PMF, using windowing samples of only 20 ns that lead to an inaccurate description of the F 1 → F 2/3 transition path. In fact, inspection of the φψ normalized average occupation density of visited φψ states reconstructed from all umbrella sampling windows shows that a lack of sampling of the F 2 well is evident, with the path seeming to hop discontinuously from the F 1 to F 3 wells (Figure ).