Area per lipid (APL) is a fundamental parameter describing the molecular packing of lipid bilayers and can reach stable values on a 100 nanosecond timescale in MD simulations. As shown in Fig. , APL gradually decreased in the process of adding more CaCl 2 to the system (Fig. ), a phenomenon that is more signiicant in the mixed POPC/POPS membrane. When the Ca 2+ concentration exceeded 200 mM, the change in APL became less pronounced. It appears that the effect of Ca 2+ on the packing of membranes containing negatively charged PS is more signiicant. With saturated CaCl 2 (200 mM), the APL was reduced by approximately 4.2% for the pure POPC bilayer (from ∼0.72 nm 2 to ∼0.69 nm 2 ) and 23.8% (from ∼0.88 nm 2 to ∼0.67 nm 2 ) for the mixed POPC/POPS bilayer. The reduction in APL was accompanied by an increase in bilayer thickness (Fig. ), which was also more signiicant in the mixed POPC/POPS bilayer. There was no signiicant difference in the APL and membrane thickness when using two different Ca 2+ models, indicating that the observed impact of Ca 2+ on the overall membrane size was rather robust and independent of the speciic Ca 2+ model used for simulations. We also examined the structural properties of the lipid tails by analyzing the acyl chain C±H bond order parameters (Eq. ( ), Fig. ). Evidently, the lipid tails in the mixed POPC/POPS bilayer systems became more ordered with increasing concentrations of Ca 2+ ions, which is consistent with previous indings, and the inluence became less signiicant when the concentration of Ca 2+ exceeded 200 mM. In contrast, the changes in the order parameters in the pure POPC bilayer systems were minor in all the simulations. The two Ca 2+ models showed consistent effects on the acyl chain order parameters as well.