When applied to NcLPMO9D with its active site exposed to solvent (i.e. the monomeric structure), the VM2 method, which performs extensive molecular conformational searching and provides a total free energy via a Boltzmann weighted ensemble of the resultant low energy conformers, predicts that both "inward" and "outward" His157 confirmations are populated. Focusing only on the two lowest energy conformers found by VM2 (Fig. ), the first conformer is "inward" and the second conformer is "outward" with a free energy difference of only 0.11 kcal mol -1 between them. A representative structure from these calculations with His157 in the outward conformation was used as the starting model of the MD simulations previously described. Furthermore, summing the probabilities of each type of conformation populated, so that 99.0% of the total population is included, His157 has a predicted 3:1 "inward" to "outward" probability distribution. In contrast, when applied to the NcLPMO9D dimer, as per the crystal structure (Fig. ), VM2 finds only conformations where His157 remains fully "inward". This is despite VM2's ability to drive molecular conformations over large energy barriers, as well as a free energy window set at 10 kcal mol -1 for retaining minima found by the conformational search. This result strongly supports preclusion of movement of His157 to an "outward" conformation due to the barricaded active site present in the crystal structure. An estimate of the barrier between the inward and outward conformation was obtained using a combination of the MD simulations and VM2 calculations. Selected conformation ranging between the two extremes were selected from MD simulations and the relative energy were obtained using VM2 calculations. These calculations suggest that the barrier is about 11.0 kcal/mol from the inward to outward conformation, and about 12.1 kcal/mol from the outward to the inward conformation. More details are available in the SI.