(A) Wildtype PLY (PLYWT) and PLYD168A form rings on cholesterol-containing liposomes. PLYWT lyses the majority of liposomes, while PLYD168Aleaves them mostly intact. Lipid-filled rings with a narrow rim (blue arrows) are prepores, while rings with a wider rim that do not contain lipid (red arrows) are pores. Slits (green arrows) and arcs (yellow arrows) are observed occasionally, but mostly PLY forms complete rings. Mutant PLYD168A prepores detach easily from the liposomes due to reduced binding affinity, and then break into fragments. Curves indicate the hemolytic activity of PLYWT, PLYD168A, and PLYΔ146/147. PLYΔ146/147 is inactive, in line with the inability of this mutant to form oligomers on cholesterol-containing liposomes. Scale bar: 50 nm.