Note that any asymmetries between the two lealets could lead to spontaneous curvature of the membrane. Three types of asymmetries have been studied: (1) compositional asymmetry Ð one of the lealets has a different lipid composition or different ratios of lipid compositions than the other; (2) number asymmetry Ð the two lealets have different numbers of lipids; (3) environmental asymmetry Ð for example, the headgroups of the lipids in the two lealets face different Ca 2+ concentrations. The spontaneous curvature caused by differential stress as a result of number asymmetry (2) has been well characterized in reference. In this paper, we studied both the compositional asymmetry (1) and environmental asymmetry (3), but neglected the number asymmetry by intentionally equalizing the number of lipid molecules in the two lealets. As shown in Fig. , in the absence of Ca 2+ concentration, compositional asymmetry alone could induce a spontaneous curvature c 1 0 ≃ 0.04 nm -1 for the lower bilayer and c 2 0 ≃ -0.06 nm -1 for the upper bilayer. Here we chose the sign convention that a negative spontaneous curvature tended to bend away from the middle water layer while a positive spontaneous curvatured tended to bend towards the middle water layer. The addition of Ca 2+ ions signiicantly enhanced the upper bilayer's tendency to bend away from the middle water layer, and could reach up to c 2 0 = -0.3 nm -1 at high centration of Ca 2+ ions. While for the lower bilayer, the presence of Ca 2+ ions was able to reverse its bending direction, from towards the middle water layer to away from it. Combining the two results, we inferred that Ca 2+ ions near a membrane tended to bend the membrane away from the Ca 2+ ions. To verify that our observation was not caused by the speciic double-membrane simulation setup, we calculated the curvature of another double membrane system, where the lower membrane is upside down, treated with 200 mM Ca 2+ (Fig. ). We found that Ca 2+ could still induce a negature curvature for the lower membrane (c 1 0 ≃ -0.16 nm -1 ), which was qualitatively aligned with those of the original simulation system (Fig. ). Therefore, although the curvature values may vary depending on the speciic simulation setup, the bending directions of the membranes are always consistent.