Where possible, individual domains of the PLY x-ray structure (van Pee et al., 2016) were moved manually into the 4.5 Å cryoEM map as rigid bodies. Rebuilding of domains that refolded upon membrane insertion and readjustment of secondary structure elements and sidechains within domains yielded an atomic model of the pore complex (Figure 1). Comparison to the soluble form (Figure 2A,B) revealed a complete reorganization of the toxin upon membrane insertion. Of the four protein domains D1-D4 in the x-ray structure, D1 and D4 fitted the map of the pore complex with minimal modifications (Figure 2—figure supplements 1–3, Video 1). In the membrane-inserted form, the loop linking D1 to D2 refolds into a helix (α3a) at the interface between the rearranged domains D1, D2 and D3 (Figures 2A,E, 3 and 4; Figure 2—figure supplement 4). In D4, the highly conserved undecapeptide loop (Figure 3) that renders PLY cholesterol-specific (Soltani et al., 2007b) was shifted by up to ~9 Å into the cryoEM map (Figure 2—figure supplements 2 and 3, Video 1). The undecapeptide loops of adjacent protomers in the pore complex are located in one plane on the outer membrane surface of the target cell, where they interact closely with the lipid head groups (Figures 1B and 2A,C). In the linear rows of soluble PLY monomers that are found in the PLY crystal structures (Lawrence et al., 2015; Marshall et al., 2015; van Pee et al., 2016), the distance between the loops of neighbouring monomers is ~14 Å. In the pore complex this distance decreases to 4–5 Å, which enables an interaction of the loop that connects β-strands 18 and 19 (β18/19) in D4 with the β22/23 loop in D4 of the next monomer along the ring (Figure 2—figure supplements 2 and 3, Video 1). The close proximity of loop β18/19 that contains Asp403, Thr405, and His407 of one monomer to loop β18/19 and the uncedapeptide containing Trp433 of the adjacent monomer suggests a critical role of these D4 loops not only in receptor recognition, but also in oligomer formation.10.7554/eLife.23644.006Figure 2.Soluble and membrane-inserted PLY monomer.One subunit of membrane-inserted form of PLY in the cryoEM structure (A) and x-ray structure of soluble PLY (van Pee et al., 2016) (B) seen from the side (left) and from the pore center (right). Both helix bundles (HB1 and HB2, cyan) in the PLY monomer refold to form two long, membrane-spanning β-hairpins (HP1 and HP2). The upper end of the β-hairpins is sandwiched between a helix-turn-helix motif (HTH, green) on the inside and helix α3a (red) of the next monomer on the outside of the pore. One of the helices is a refolded β-strand of D3 (green). Helix α3a is the refolded linker that connects D2 to D1 in the soluble monomer. (C) D4 contains the conserved undecapetide (green sticks) that confers cholesterol specificity to PLY. The peptide includes three resolved Trp sidechains, one of which interacts with Thr405 in the adjacent monomer. (D) Cross section through a segment of the 168-strand β−barrel with resolved bulky sidechains. The two β-hairpins of one monomer are labelled. (E) Helix α3a interacts with HP1 and D1 of the neighbouring monomer, stabilizing the pore complex.DOI: http://dx.doi.org/10.7554/eLife.23644.00610.7554/eLife.23644.007Figure 2—figure supplement 1.Stereo view of domain 1 presented as ribbon-and-stick model.Selected large sidechains are shown as sticks. The map is contoured at 7.0 and 9.0 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.00710.7554/eLife.23644.008Figure 2—figure supplement 2.Superposition of domain 4 of the PLY monomer in the crystal structure (blue) and in the pore complex (green), viewed from the pore center.Residues of the undecapeptide and selected residues in other loops are shown as sticks. The map is contoured at 6.5 and 7.5 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.00810.7554/eLife.23644.009Figure 2—figure supplement 3.Stereo view of domain 4 in the pore complex with potential inter- and intramolecular interactions of loops viewed from two sides.The undecapeptide is green and selected residues are shown as sticks. The closest distance between the loops from different monomers is 4–5 Å. The map is contoured at 6.0 and 7.5 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.00910.7554/eLife.23644.010Figure 2—figure supplement 4.Stereo view of domain 3 with the upper part of the β-barrel and the new helix-turn-helix motif (HTH) that forms the α-barrel inside the pore.The new helix α3a (red) from the neighboring monomer is on the right. Hydrophobic residues at the interface between the β-sheet and HTH and intermolecular interactions to the next helix α3a are shown as stick models. The map is contoured at 6.5 and 8 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.01010.7554/eLife.23644.011Figure 2—figure supplement 5.Stereo view of domain 2 seen along the ring axis.The main chain is presented as cartoon and the side chains as sticks. The map is contoured at 7.0 and 9.0 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.01110.7554/eLife.23644.012Figure 3.Sequence alignment of selected cholesterol-dependent cytolysins.Elements of secondary structure are shown for the crystal structure of soluble PLY (above) and for the cyroEM structure of the pore complex (below). Colors of the secondary structure elements and residue numbers correspond to the PLY crystal structure (Figure 2B; pdb code 5aod). Asterisks indicate conserved residues.DOI: http://dx.doi.org/10.7554/eLife.23644.01210.7554/eLife.23644.013Figure 4.Intermolecular interactions in the 4.5 Å cryoEM map of the PLY pore complex.Slices parallel (A–C) and perpendicular (D–F) to the membrane reveal interactions of secondary structure elements between adjacent monomers in the pore complex. Section planes are indicated on top. Note that the scale of sections D to F is 50% larger to show molecular detail more clearly. The β5-α4-β6 region in domain D1 and the long membrane-parallel helix α5 (red box in A and D) alternate around the top of the pore complex. On the outside, the 168-strand β-barrel is flanked by helix α3a (orange box in B and D) and by the α-barrel of the helix-turn-helix motifs (green box in B and F) on the inside. The 4-strand β-sheets of domain D4 (blue box in C and E) are offset against D4 of the next monomer, forming a ring of 8-strand β-sheets. The 85 Å-long β-strands forming the 168-strand β-barrel are clearly resolved (cyan box in C and E).DOI: http://dx.doi.org/10.7554/eLife.23644.013Video 1.Superposition of domain 4 of the PLY monomer from the crystal structure (blue) on domain 4 of the PLY monomer in the pore complex (green) rotated around an axis perpendicular to the membrane.Residues of the undecapeptide and selected residues in other loops are shown as sticks.DOI: http://dx.doi.org/10.7554/eLife.23644.01410.7554/eLife.23644.014