On the other hand, our improved DFTB3 Hamiltonian (curve in blue in Figure ) provided an activation free energy of 16.8 kcal/mol that was in perfect agreement with previous theoretical reports for DHFR from Escherichia coli, and experimental data (14.2-14.3 kcal/mol). It is worth-noting that our calculations do not account for nuclear quantum effects, which are expected to lower the activation barrier and improve alignment with experimental results. 70