To understand the functional role of individual sidechains in oligomerization, prepore and pore formation, we mutated Asp168 that, as the structure suggests, might be involved in forming a salt bridge between adjacent subunits to alanine (PLYD168A). We also deleted Ala146 and Arg147 (PLYΔ146/147) in the loop that would clash with the last β-strand of the central β-sheet. The resulting mutants were characterized by negative-stain EM (Figure 7A), hemolytic activity assays (Figure 7B), and x-ray crystallography (Figure 8). X-ray data were collected to 2.45 and 2.5 Å resolution from crystals of PLYD168A and PLYΔ146/147 (Table 1). The structures were solved by molecular replacement with the x-ray structure of PLYWT (van Pee et al., 2016). As in PLYWT, the mutant monomers crystallized in rows. Superposition of both structures on PLYWT showed no significant overall differences, although a detailed comparison indicated small changes in domain D2, helix bundle HB2 and in two of the D4 loops (Figure 8). Around the mutated residue in PLYD168A differences were restricted to sidechain orientations, while the deletion of two residues in PLYΔ146/147 caused significant conformational changes in the loop connecting helix α5 to strand β7 of the central β-sheet. This deletion also caused a slight shift of helix α5 towards D3 (Figure 8).10.7554/eLife.23644.019Figure 8.Location of functionally important PLY residues.(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).DOI: http://dx.doi.org/10.7554/eLife.23644.01910.7554/eLife.23644.020Table 1.Data collection and refinement statistics.DOI: http://dx.doi.org/10.7554/eLife.23644.020PLYD168A (pdb-id 5aoe)PLYΔ146/147 (pdb-id 5aof)Data collectionBeamlinePXII @ Swiss Light Source x10saResolution (Å)40–2.5 (2.6–2.5)40–2.45 (2.55–2.45)Wavelength (Å)0.97860.978Space groupP21P 21 21 21Cell dimensionsa, b, c (Å)160.86 24.66 208.3524.73 163.5 207.8α, β, γ (°)90, 90.26, 9090, 90, 90Total reflections316531 (30235)137706 (13089)Unique reflections59222 (5685)29722 (2937)Multiplicity5.3 (5.3)4.6 (4.5)Completeness (%)99 (100)91 (94)Mean I / σI8.1 (1.4)9.1 (1.2)Wilson B-factor46.7545.05Rpim0.08 (0.593)0.07 (0.607)CC*0.998 (0.887)0.999 (0.792)RefinementReflections used in refinement59207 (5683)29718 (2936)Reflections in test set2962 (285)1486 (147)Rwork/Rfree (%)22.25/24.98 (32.9/33.7)20.87/23.44 (33.23/34.37)CC(work)/CC(free)0.946/0.934 (0.769/0.737)0.955/0.930 (0.626/0.567)Average B-Factor (Å2)72.564.3No. atoms in AU78784008Protein77243856Water154152r.m.s. deviations:Bond lengths (Å)0.0030.03Bond angles (°)0.720.69Ramachandran favored (%)9696Ramachandran allowed (%)3.63.1Ramachandran outliers (%)0.50.8Note: Values for the highest resolution shell are shown in parentheses.