Ccr's QM region was reduced compared to our previous study as the carboxylation reaction was not studied, making it comparable to the model used in the initial benchmark calculations (see Figure ). The link atom approach, as implemented in the sander program of the AmberTools18 suite, was employed to saturate missing valences at the QM/MM interface, summing to a total of 48 QM atoms (two link atoms). Initial QM/MM MD simulations were performed in sander using the DFTB3 semiempirical method's (3OB-3-1 parameter set), while the MM region was treated with the CHARMM36 force field and parameters derived for NADPH cofactor and crotonyl-CoA substrate. 41 The adaptive string method (ASM) was used to study the hydride transfer reaction in Ccr. Our implementation of the ASM approach in sander has been used extensively for the study of several enzymatic reactions. This allows finding the minimum free energy path (MFEP) between two states, in this case, the reactants and the enolate intermediate, within a space of collective variables (see Figure ) and generating the final free energy profile for the reaction under study. The validation of the selected sets of DFTB3 parameters was finally completed through free energy calculations using the ASM approach. In the case of the potential of mean force (PMF) profiles obtained at the DFTB3/CHARMM36 level with modified parameters, corrections were made at the M06-2X/def2-TZVP/CHARMM36 level, as we report in our previous work, to evaluate the new DFTB3 parameter sets' performance in the predictions of the free energy surface (FES). Full free energy calculations at the M06-2X/def2-SVP/CHARMM36 level were also carried out and served as energetic and structural reference to the semiempirical calculations described above.