Altogether, we were able to add new useful functionalities to the PCVA approach and we verified its success in systematic QM region construction. We set the starting point for new application cases especially for non-protein systems and further developments by assessing important difficulties and problems in QM region construction not only occurring for PCVA. In particular, an approach must be developed which helps to systematically calculate the ideal QM region size for any system. Furthermore, still all approaches considered here exclusively focus on the electrostatic effect of amino acids and by that might miss important non-electrostatic interactions. This problem should be addressed in the future to further improve the setup process of QM/MM calculations.