To investigate the structures of the PLY prepore and pore complex in native membranes prior to detergent solubilisation, we incubated cholesterol-containing liposomes with purified PLY and generated 3D volumes by electron cryo-tomography (cryoET). The liposomes were studded with numerous ring-shaped complexes of 290 to 360 Å outer diameter (Figure 6A). Assemblies were identified as PLY pores or prepores by the absence or presence of a lipid bilayer within the ring (Figure 7A). Pore and prepore complexes were classified independently and processed by subtomogram averaging (Figure 6B). The subtomogram average map of the prepore complex at a resolution of 22 Å (Figure 6C) had an outer diameter of 320 Å and accommodated 34 subunits (Figure 6D). Of the available PLY crystal structures (Lawrence et al., 2015; Marshall et al., 2015; van Pee et al., 2016), the model 5cr6 (Marshall et al., 2015) fitted the map best (Figure 6—figure supplement 1), indicating that in this structure the soluble toxin is in the pre-pore state. The subtomogram average map of the pore complex at 27 Å resolution (Figure 6C) indicated a slightly larger diameter of around 350 Å, but likewise consisted of 34 subunits (Figure 6E). The cross-section profile resembled that of the high-resolution cryoEM map of the pore complex closely (Figure 1), indicating that solubilisation with detergent or amphipol does not change the structure of the membrane form significantly. The model of membrane-inserted PLY fitted the subtomogram average of the pore complex without significant adjustment, including the 20° inclination of the long β-hairpins relative to the membrane normal.10.7554/eLife.23644.016Figure 6.CryoET of PLY prepores and pores.(A) PLY assembles into prepores and pores upon incubation with cholesterol-containing liposomes. Scale bar, 50 nm. (B) Sections through subtomogram average volumes of the prepore (top) and pore complex (below). Left panel: sections perpendicular to the membrane; right panel: sections parallel to the membrane. Colors indicate section planes. (C) Fourier shell correlation for subtomogram averages indicate 22 Å resolution for the prepore and 27 Å for the pore at FSC0.5 or 20 Å and 21 Å resolution at at FSC0.3. Oblique view (D) and cross section (E) of PLY prepore (left) and pore (right). Both maps accommodate 34 PLY monomers. The prepore map was fitted with the crystal structure of the soluble PLY monomer (Marshall et al., 2015), and the pore map with the cryoEM structure of the pore monomer (Figure 2A). PLY domains are red (D1), yellow (D2), green/cyan (D3) and blue (D4). The lipid bilayer is continuous in the prepore complex, but absent in the pore complex.DOI: http://dx.doi.org/10.7554/eLife.23644.01610.7554/eLife.23644.017Figure 6—figure supplement 1.CryoET map of the PLY prepore with rigid-body fitted x-ray structures of the water-soluble toxin that forms rows in the 3D crystals.PLY monomers of 5AOD (green) are straight and protrude by 10 Å at the top of the map. Monomers of 5CR6 (red) are bent at the hinge between domain 2 and 4 (blue arrow) and match the map volume closely, indicating that this structure represents the pre-pore form of PLY.DOI: http://dx.doi.org/10.7554/eLife.23644.01710.7554/eLife.23644.018Figure 7.Membrane binding and hemolytic activity of PLY .(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.DOI: http://dx.doi.org/10.7554/eLife.23644.018