QM subsystems consisted of 217-221 atoms for hBChE-DEP scans, 173-220 for hAChE in different stages, 171-230 for hBChE in different stages, and 171-173 for the tcAChE(-NV) free form. Free forms lacked a portion of the oxyanion hole, otherwise all other stages and systems shared the same composition that differed only by the nature of the ligand or adduct (Figure ). To prevent link atom hyperpolarization, a "charge shifting" scheme was used. The effect of non-integer charge of the QM system on PME was alleviated with the Amber scheme as discussed before. The QM region was described with DFTB3 49 with 3ob-3-1 parameter set similarly to what was used before in various enzymological tasks. Additionally, D4 dispersion correction was utilized. All reported simulations were performed with periodic boundary conditions and full PME treatment for QM/MM electrostatics.