By contrast, the structure of D3 changes entirely upon membrane insertion. In the soluble toxin, the central, five-stranded β-sheet in D3 is flanked by two bundles of short α-helices (Figure 2B). In the pore complex, both bundles refold into four 85 Å β-strands, which form two parallel β-hairpins that insert into and traverse the lipid bilayer (Figure 2A). The β-hairpins of neighbouring subunits coalesce into one extensive, 168-strand β-barrel with an inner diameter of 260 Å (Figure 1B). The new β-strands can be traced unambiguously, because they are straight, apparently rigid and continuous with four β-strands in the D3 x-ray structure that are preserved in the membrane form. The two new, long β-hairpins are inclined by 20° relative to the membrane normal, in good agreement with predictions for perfringolysin O on the basis of cysteine crosslinking experiments (Sato et al., 2013). The chain trace in the refolded domain is confirmed by the positions of bulky densities for large sidechains in the β-barrel (Figure 2D; Figure 2—figure supplement 4). While five of the six helices in bundles HB1 and HB2 refold into β-strands in the pore complex, one remains intact. Conversely, the loop and fifth β-strand in the central β-sheet of D3 refold into a helix (Figure 2A,B). Together, the two α-helices form a helix-turn-helix (HTH) motif (Figure 2—figure supplement 4). In the ring, the HTH motifs of the 42 subunits line up in one plane on the exoplasmic side of the molecule and form an α-barrel inside the β-barrel (Figures 1B and 4), restricting the pore diameter locally to 250 Å (Figure 1B).