Conformational and functional analysis of α-HL oligomers in an instant hemolysis environment. (A) Compromised hemolysis of RBC at a lower temperature (10°C) with different toxin concentrations as compared to hemolysis at 37°C. (B) Membrane rigidity was measured between RBCs kept at 37°C (for 15 min) and 4°C (for 1 h) using a solvatochromic dye Nile red (emission range between 585 and 610 nm showed in red color, and 525 and 565 nm showed in green color). (C) CD analysis of toxin at different temperatures with or without oligomerizing agents (POPC-LUVs). (D) Cryo-EM micrograph showing toxin particles, and 2D class averages showing heptameric and octameric states. (E) 2D class averages of different sizes of arcs are highlighted in different square boxes (blue—4-mer or tetramer; white—5-mer or pentamer; and orange—6-mer or hexamer). (F) 3D reconstruction (top and side views, in gray color) of the pentameric arc-like intermediate species. CD, circular dichroism.