Earlier cryoEM work, AFM and FRET studies have described the vertical height change of the prepore upon membrane insertion in terms of a collapse or unfolding of D2 (Czajkowsky et al., 2004; Ramachandran et al., 2005; Tilley et al., 2005). Our atomic model of the pore complex now shows that this change is due to a rigid-body rotation of D2, in which the structure of the domain remains intact. Time-lapse AFM of PLY and listeriolysin (LLO) on planar membranes indicated that the vertical height change is not directly linked to pore formation (Mulvihill et al., 2015; van Pee et al., 2016), in line with a mechanism that involves two different prepore states that we refer to as the early and late prepore. Our model of the late prepore monomer (Figure 9) is an intermediate between the x-ray structure of the soluble form and the cryoEM structure of the membrane-inserted form. The 5-stranded central β-sheet in the crystal structure of D3 fits into the cryoET map of the early prepore complex without any modification, but the late prepore model indicates a potential steric clash between the short β-strand in the central β-sheet of D3 and the adjacent monomer. The pivotal role of the loop that contains residues 145–147 connecting helix α5 of D1 to β7 of β-hairpin one is underlined by mutant PLYΔ146/147 that was completely inactive (Kirkham et al., 2006) and did not even bind to liposomes (Figure 7A). The detachment of PLYD168A rings indicates that the mutant does not insert into the membrane as easily as PLYWT to form pores. We have shown by time-lapse AFM that pore formation is irreversible (van Pee et al., 2016). Therefore the rings that become detached from the membrane are prepores, not pores.10.7554/eLife.23644.021Figure 9.Mechanism of membrane insertion and pore formation.Stepwise conformational changes of PLY toxin during pore formation shown for one monomer (above, side view) and for three neighbouring monomers (below, view from the pore centre). In the first step, soluble PLY monomers attach to the surface of cholesterol-containing cell membranes via the conserved D4 undecapeptide (Figure 2C) to form circular oligomers of the early prepore. A 90° rotation of D2 moves D1 and D3 towards the membrane in the late prepore. Helix bundles HB1 and HB2 are poised above the membrane surface to refold into 85 Å β-hairpins HP1 and HP2. In the final step of pore formation, both hairpins traverse the hydrophobic membrane core and assemble into a 168-strand, 260 Å β-barrel. Reorganization of the PLY monomer exposes numerous charges on the inside of the β-barrel (Figure 4) that would destabilize the lipid bilayer and repel membrane lipids, resulting in pore opening and cell lysis.DOI: http://dx.doi.org/10.7554/eLife.23644.021