Note, as described in the methods section, 6 distance restraints were applied with the neighboring atoms to keep Cu in the active-site. The MD simulation based on 5UFV also used restraints between Cu and the equivalent residues, which excludes the possibility that the lack of interconversion between the inward and outward conformation in MD based on our structure is due to the use of distance restraints. To further explore the conformational flexibility, we performed another MD simulation starting from outward conformation (see details below). In this MD simulation, His157 was able to interconvert to the inward conformation at the microsecond timescale (only twice), confirming our hypothesis of dynamic interconversion of His157. However, once fully in the inward conformation, even in this MD simulation His157 stayed locked in that position. It is interesting that the two related LPMOs show difference in the rates of His157 (and equivalent His161) conformation interconversion, which could also have a functional role possibly associated with sampling of certain conformational events required in the catalytic cycle as has been observed in several other enzyme systems. To further probe the extent of such local flexibility and the associated energetics in our own structure, we carried out 2 nd -generation mining minima (VM2) free energy calculations, and compared the predicted thermodynamically accessible NcLPMO9D His157 conformations found when the active site is solvent exposed to those found when the active site is in the crystal structure with binding-face to binding-face packing.