Cf 400

T4SS_3-10/B4-His6 or T4SS_3-10/His6-B6 complexes were applied onto a glow-discharged carbon coated grid (CF-400, Electron Microscopy Sciences). After 1 minute, excess of liquid was blotted, and the grid was washed on a drop of cold purification buffer (50 mM Tris pH8, 200 mM NaCl, 1 mM TCEP, 0.1 % w/v digitonin, 0.06 % w/v DM-NPG) containing 50 mM imidazole, quickly blotted and deposited on a second drop of the same buffer in the presence of 5 nM nanogold beads (Nanoprobes). After 2 minutes, the grid was rinsed sequentially for 20 seconds with: 1 drop of purification buffer, 1 drop of the same buffer without detergent and 3 drops of 2 % uranyl acetate. Incubation was for 1 minute. Images were collected on a Tecnai T12 BioTWIN LaB6 microscope operating at a voltage of 120 kV. Particles were selected manually using EMAN.

Fresh purified complex (4 μl at ~0.01 mg/ml) was applied to glow-discharged carbon-coated grids (CF-400, Electron Microscopy Sciences). The sample was negatively stained with 2 % uranyl acetate and visualised in a FEI Tecnai T12 BioTWIN LaB6 microscope operating at a voltage of 120 kV. Images were recorded on a FEI Eagle 4K×4K CCD camera under low dose conditions (~25 e/Ų) at a magnification of circa 44,000 (3.4 Å/pixel) and a defocus range of 1-2.5 μm. The CTF parameters were assessed from entire image frames using CTFFIND3. Phase flipping was carried out using SPIDER and applied to entire frames. A total of 1284 particles were selected manually from CTF-corrected micrographs using BOXER (EMAN2). Boxed images were normalised, band-pass filtered and centered. They were then subjected to a reference-free classification. Iterations of MRA using representative views of the complex and MSA were performed in IMAGIC until the classification of the dataset stabilized. The final classes were generated with approximately 20 images per class.

Negative stain EM was used to monitor in-solution oligomerization states of CsgG, CsgG_C1S and CsgE. CsgE, CsgG_C1S and amphipol-bound CsgG were adjusted to a concentration of 0.05 mg ml⁻¹ and applied to glow-discharged carbon-coated copper grids (CF-400; Electron Microscopy Sciences). After 1 min incubation, samples were blotted, then washed and stained in 2% uranyl acetate. Images were collected on a Tecnai T12 BioTWIN LaB6 microscope operating at a voltage of 120 kV, at a nominal magnification of ×49,000 and defocus between 800 and 2,000 nm. Contrast transfer function (CTF), phase flipping and particle selection were performed as described for cryo-EM. For membrane-extracted CsgG, octadecameric particles (1,780 in all) were analysed separately from nonamers and top views. For purified CsgE a total of 2,452 particles were analysed. In all cases, after normalization and centring, images were classified using IMAGIC-4D as described in the cryo-EM section. The best classes corresponding to characteristic views were selected for each set of particles. Symmetry determination of CsgG top views was performed using the best class averages with roughly 20 images per class. The rotational autocorrelation function was calculated using IMAGIC and plotted.