Structural analysis of α-HL heptamer in the presence of RBC environment. (A) Cryo-EM micrograph of α-HL oligomers in the RBC environment at a magnification of 54 k× and zoomed view showed circular toxins (white arrowhead). (B) 2D class averages showed disk-shaped top views of toxins and mushroom-shaped side views. (C) Cryo-EM maps of α-HL heptameric pore structure at 3.1 Å resolution; each protomer is shown in different colors (from left, lime green, golden rod, cyan, hot pink, olive drab, dodger blue, medium orchid). The cross-section of the map showed a pore channel and extra density (gray color) corresponding to the membrane surrounding the barrel and the lower part of the rim domain. (D) Particle distribution after 3D classification shown in a bar plot (heptameric pore species in red color and heptameric prepore in purple color). (E) Mass spectrometric analysis of lipids associated with the toxin. Left panel showing the spectrum of lipids present in the pellet fraction, and the right panel showing the spectrum of lipids present in the supernatant fraction. (F) PC head group fitting in the EM density map (gray color). Seven PC head groups (red color) were fitted symmetrically at the topmost density in between the rim and stem domain. The interacting amino acids (golden color) from the rim and stem domains with the lipid are Y112, H144, Y148, W179, and R200. (G) Flow cytometry analysis of PI-positive HL-60 cells after incubation with mutant toxins (100 nM concentration). Data are shown as the mean ± SD of triplicate measurements (n = 3 biological triplicates).