Figure 1.

Lipid-5 insertion algorithm. (A) Different steps involved in the insertion algorithm. The first step involves an initial equilibrated bilayer system as input to the algorithm. Here, the initial system consists of DSPC and cholesterol lipids, shown in blue and orange, respectively. The headgroups of DSPC are shown in orange circles, where the hydroxyl group of cholesterol is represented in red dots. The first step is followed by random selection of a predetermined number of DSPC molecules (shown in red). A series of repulsive grid potentials (shown in faded gray) are generated at the place of selected DSPCs, generating enough space to insert ionizable lipids. Lipid-5 (shown in gray) is inserted into the bilayer followed by gradual removal of the grid potentials. Finally we generate a new bilayer containing the desired proportion of the ionizable lipid. (B) Lipid-5 molecular structure. The nitrogen in Lipid-5 is titratable and can change its protonation depending on the buffer pH. Carbon, oxygen, and nitrogen atoms are shown in gray, red, and blue, respectively.