(A) Ala146 and Arg147 in the loop that induces refolding of the last β-strand in the central D3 β-sheet into helix α13a in the late prepore and pore complex. Deletion of both residues renders the toxin inactive. (B) In the pore complex, Asp168 near the end of one long trans-membrane β-hairpin (HP1) forms a salt bridge with Lys271 in the other trans-membrane β-hairpin (HP2) of the adjacent monomer. Replacing Asp168 by alanine inhibits membrane insertion. (C) α-carbon traces in the x-ray structures of PLYWT (pdb 5aod), PLYΔ146/147 (pdb 5aof), and PLYD168A (pdb 5aoe). Minor differences between wildtype and mutant structures are visible in the loop regions of D4 (blue arrows); D2 (yellow arrows) and HB2 (green arrows). In PLYΔ146/147, one loop connecting D1 to D3 is also slightly different (red arrows).