Cryo-EM sample preparation for α-HL–treated RBCs and cryo-EM analysis of oligomeric states of α-HL obtained from posthemolytic RBC–toxin complex. (A) Sample preparation procedure of α-HL–treated RBCs (created with BioRender.com). SDS-PAGE gel showing lanes corresponding to untreated RBC pellet—lane 1; untreated RBC supernatant—lane 2; toxin-treated RBC pellet—lane 3; toxin-treated RBC supernatant—lane 4; and protein marker—lane 5. (B) NS-TEM analysis of the supernatant fraction of α-HL–treated lysed RBCs. An enlarged view of the white squared region is shown, the oligomers are encircled in yellow color, and the 2D class averages of those particles are shown. (C) Cryo-EM data processing pipeline of α-HL oligomers present in the posthemolytic condition. The extra membrane density is shown in yellow, and the map in gray. (D) Atomic model of heptameric pore structure (in cyan). (E) Comparison of pore structure solved in the presence of RBCs (cyan) with the pore structure solved in the presence of detergent (pink). The shifts in S262 and G180 residues in the rim domain are 2.4 Å and ~2 Å, respectively.