Figure 3. Cryo-EM structure of the tridecameric XaxAB pore complex.
(a) Cryo-EM density map of tridecameric XaxAB pores shown as top, side and bottom view. XaxA and XaxB are colored in green and yellow, respectively. (b) Ribbon representation of the atomic model of XaxAB. Colors shown as in (a).
Figure 3—figure supplement 1. Pore formation of XaxAB induced by different detergents and analyzed by negative stain EM.
(a-f) Representative micrographs of negatively stained XaxAB pore complexes formed by incubation with different detergents, in particular n-Octyl-β-D-glucopyranoside (OG) (a), Lauryl maltose neopentyl glycol (LMNG) (b), 6-Cyclohexyl-1-hexyl-β-D-maltoside (Cymal-6) (c), 7-Cyclohexyl-1-hexyl-β-D-maltoside (Cymal-7) (d), 3-[(3-Cholamidopropyl)-dimethylammonio]−1-propane sulfonate] (CHAPS) (e), and Digitonin (f). Scale bars, 50 nm.
Figure 3—figure supplement 2. Analytical size exclusion chromatography and negative stain EM of XaxAB in Cymal-6 and amphipols.
(a) Size exclusion chromatography profile of XaxAB after incubation overnight with Cymal-6. (b) Representative electron micrograph of negatively stained XaxAB after incubation overnight with Cymal-6 (c). (c–d) Size exclusion profile (c) and representative electron micrograph (d) of the negatively stained XaxAB pore complex in amphipols. Scale bars, 50 nm. Arrows indicate fractions analyzed by negative stain electron microscopy.
Figure 3—figure supplement 3. 2-D class averages of XaxAB pores with different numbers of subunits.
Representative class averages of top views showing XaxAB pores with 12, 13, 14, and 15 subunits are highlighted in yellow, red, green and blue, respectively. Scale bar, 10 nm.
Figure 3—figure supplement 4. Single particle processing workflow of XaxAB structure determination.
During 3-D refinement a mask excluding the amphipol ring is applied to improve alignment of the particles during 3-D reconstruction.
Figure 3—figure supplement 5. Cryo-EM structure of tridecameric XaxAB.
(a-b) Representative digital micrograph (a) and selected 2-D class averages (b) of the XaxAB toxin complex embedded in vitrified ice. Scale bars, 50 nm (a), and 10 nm (b). (c) Ab initio 3-D reconstruction generated with RVIPER. (d) Angular distribution of the particles. (e) Cryo-EM density map of XaxAB colored according to the local resolution. (f) Fourier Shell Correlation (FSC) curves between maps from two independently refined half data sets (black) and the final map versus the atomic model (grey). The 0.143 criterion shows an average resolution of 4 Å. (g) Representative regions of the density with fitted atomic models of XaxA (green) and XaxB (yellow).
Figure 3—figure supplement 6. Transmembrane domains of XaxAB embedded in amphipols.
The amphipol density is shown in transparent grey at a lower threshold to allow better visualization. Protomers of XaxA and XaxB are depicted in green and yellow, respectively.
Figure 3—figure supplement 7. Biophysical properties of the XaxAB pore.
(a-c) Top and side views on the surface and the inside of the pore complex, showing the conservation of residues (a), the surface electrostatic Coulomb potential at pH 7.5 (b), and the surface hydrophobicity (c). Conserved residues are shown in magenta, positively and negatively charged surfaces are colored in blue and red, respectively and hydrophobic patches are depicted in orange.
Figure 3—figure supplement 8. Amino acid sequence alignment and conservation of the transmembrane region of XaxA (a) and XaxB (b).
Yellow boxes highlight the conserved positively charged region in XaxA (a) and the conserved proline (P204) and glycine (G243) residues located at the hinge regions of XaxB (b).
Figure 3—figure supplement 9. Comparison of the XaxAB pore complex with other α-PFTs.
(a, b) Top (a) and side (b) views of the XaxAB, ClyA (pdb-ID: 2WCD) and FraC (pdb-ID: 4TSY) pore complexes. (c) Surface electrostatic Coulomb potential at pH 7.5 on the inside of the XaxAB, ClyA and FraC pore complexes. Positively and negatively charged surfaces are shown in blue and red, respectively. The membrane is indicated by a grey band.