Motivated by the results of a global point charge variation analysis presented in the previous section, we set out to develop a protocol of the systematic construction of the QM region that aims at minimizing the sensitivity of the QM/MM reaction energy. To this end, we consider the sensitivity of the QM/MM reaction energy with respect to variations of the point charges in single amino acids, i.e., we perform a single amino acid PCVA. The resulting protocol for systematic QM region construction is summarized in Fig. and will be described in the following.