The second approach is based on the upgraded Amber force field (UAFF) parameters for zinc-binding residues, which uses Amber force field ff14SB 34 together with modified parameters for the zincs and their coordinating residues. We combined the UAFF GRO-MACS compatible files from Macchiagodena et al. with the protein ff14SB force field and updated the missing parameters of the δ-protonated histidine dihedrals manually as suggested by Zheng. Similarly, the ffbonded.itp file given in Macchiagodena et al. for the GLZ and ASZ residues was edited to include the carbon atom (atom type CT) to which the zinc-coordinating oxygen (atom type OD1) is bonded. For editing the Gromacs ff14SB files, we followed the Gromacs instructions for adding a custom residue to a force field. Following the instructions in Macchiagodena et al., the zinc-coordinating residues have been renamed to their corresponding UAFF names in our input pdb-file. After parameterization, we converted the Gromacs-formatted topology and coordinate files into Amber format with BioSimSpace. Both RBFE and MD simulations were carried out for both the restraint approach and the UAFF method.