Together with the x-ray structure of the soluble form, our cryoEM structures of the pore and prepore explain the step-by-step process of membrane insertion and pore formation in near-atomic detail. Movies showing the transition from the soluble monomer to the pore complex via the intermediate stages of the early and late prepore illustrate the complete mechanism of pore formation (Videos 2–4). In the early prepore, PLY monomers assemble side by side into rings on the membrane surface. In the late prepore, the D3 helices are poised for re-folding immediately above membrane surface. Helix unfolding may be a stochastic yet cooperative event, such that the spontaneous transition of one monomer triggers the refolding of its neighbours in the ring, comparable to a zipper. As the 168-strand β-barrel assembles in the membrane, the charged patches on its inside surface would repel any trapped hydrophobic lipid. Most likely the lipid is pushed out of the nascent pore by successively inserted toxin monomers, rather than being ejected in the form of micelles or small vesicles, as has been proposed for suilysin (Leung et al., 2014). Upon pore formation, the membrane potential collapses, the cytoplasm leaks out and the cell dies.Video 2.Mechanism of pneumolysin pore formation.(1) Soluble PLY monomers attach to cholesterol-rich membranes by the cholesterol-binding undecapeptide (light green) of domain D4 (blue) and oligomerize into rings. For simplicity, only three ring subunits are shown. (2) Domain D2 (yellow) rotates by 90°, bringing domain D3 (green) with its two helix bundles (cyan) close to the membrane surface. (3) The helix bundles insert into the membrane and unfold into two trans-membrane 85 Å β-hairpins. β-hairpins of the 42 subunits in the ring merge into one large 168-strand β-barrel, which perforates the membrane.DOI: http://dx.doi.org/10.7554/eLife.23644.02210.7554/eLife.23644.022Video 3.Model of conformational changes in an entire ring of 42 PLY monomers from early prepore to late prepore to pore, seen from the side and from above.DOI: http://dx.doi.org/10.7554/eLife.23644.02310.7554/eLife.23644.023Video 4.Oblique view of PLY ring inserting into the membrane.DOI: http://dx.doi.org/10.7554/eLife.23644.02410.7554/eLife.23644.024