Ion selectivity in protein binding sites is of great significance to bi-ological functions.Although additive force fields have been successfully applied to various protein-related studies,it is diffi-cult to well capture the subtle metal-protein interaction for the prediction of ion selectivity,due to the remarkable polarization and charge transfer effect between the metals and the surrounding residues.Quantum mechanics-based methods are well-suited for dealing with these systems,but they are too costly to apply in a direct manner.In this work,the reference-potential method (RPM) was used to measure the selectivity for calcium and magnesium cations in the binding pocket of parvalbumin B protein by calculating the free energy change associated with this substitution reaction at an ab initio quantum mechan-ics/molecular mechanics (QM/MM) level.The alchemical transformations were performed at the molecular mechanics level,and the relative binding free energy was then corrected to the QM/MM level via thermodynamic perturbation.In this way,the free energy change at the QM/MM level for the substitution reaction was obtained without running the QM/MM simulations,thus remarkably enhancing the efficiency.In the reweighting process,we found that the selection of the QM region greatly affects the accuracy of the QM/MM method.In particular,the charge transfer effect on the free energy change of a reaction cannot be neglected.