DHFR has become a model system for enzyme-catalyzed reactions, making it an excellent choice for evaluating the new parametrization. QM/MM molecular dynamics simulations were carried out using the ASM at the DFTB3/CHARMM36 level of theory for the original and optimized DFTB3 Hamiltonian. Figure shows details on the reaction and ASM setup, as well as the free energy profiles obtained at the DFTB3/CHARMM36 level of theory (curves in blue) and the correction made using the Hamiltonian M06-2X/def2-TZVP/CHARMM36 on DFTB3 geometries (curve in red in Figure ). Similar to the case of Ccr, the free energy profile generated using DFTB3 with original 3OB-3-1 parameters severely underestimated the activation barrier and showed a minimum at the TS region, as evidenced by the corrections at higher level (see the blue and red curves in Figure ).