2.2. System Preparation. Crystal structures were obtained from the PDB and protonated using the H++ web server, for pH = 7.0, salinity of 0.15 M, and dielectric constants ε in = 10 and ε out = 80. Ligands were protonated separately using PyMOL. Except as noted below, the resulting structures were relaxed using the semiempirical GFN2-xTB model with a generalized Born/ solvent-accessible surface area (GB/SASA) implicit solvation model for water. 76 GFN2-xTB affords reasonable protein geometries as compared to crystal structures, whereas direct calculation of protein-ligand interaction energies with GFN2-xTB affords mixed results as compared to DFT calculations. Both metalloenzymes (1ZP5 and 1MMQ) proved difficult to relax using GFN2-xTB, as the Zn 2+ ion was repeatedly expelled from its binding site in numerous attempts to optimize the geometry. For these two systems, we relaxed the geometry using GFN-FF, 81 a polarizable force field designed for biological macromolecules.