Purification of the T4SS3‐10+D4 complex, single‐component deletion mutants and FLAG‐tagged T4SS3‐10+D4FLAG variant

The T4SS_3‐10+D4 complex or the single‐component deletion mutants of the T4SS_3‐10+D4 complex were purified according to the following procedure. Overnight culture of a freshly transformed TOP10 cells (Thermo Fischer) were diluted into six litre of LB medium supplemented with 100 μg/ml of carbenicillin. Cells were grown at 37°C with shaking. When the OD₆₀₀ of the cell culture reached 0.5, cells were induced by addition of 0.08% arabinose and the expression was carried on at 16°C with shaking overnight. Cells were pelleted by spinning down for 30 min at 5,000 × g and resuspended in lysis buffer: 50 mM HEPES pH = 7.6, 200 mM sodium acetate, 1 mM EDTA pH = 8.0 with a protease inhibitor cocktail tablet (Roche), 100 μg/ml lysozyme and 1 μg/ml DNaseI. After stirring of the mixture on ice for 15 min, the cells were lysed by passing them twice through an Emulsiflex C‐5. Cell debris was separated from the supernatant by spinning for 30 min at 26,712 × g. Membranes were pelleted by ultracentrifugation of the supernatant for 45 min at 95,834 × g and manually homogenized using solubilization buffer: 50 mM HEPES pH = 7.6, 200 mM sodium acetate, 1 mM EDTA pH = 8.0, 0.5% w/v n‐dodecyl‐β‐D‐maltopyranoside (DDM) (Anatrace), 0.5% digitonin (Sigma‐Aldrich), 0.05 mM n‐tetradecyl‐N,N‐dimethylamine‐N‐oxide (TDAO) (Anatrace), 2 mM tris(2‐carboxyethyl)phosphine (TCEP) (Sigma‐Aldrich) and placed on the rotary shaker at 4°C for 1 h. Insoluble materials were separated by another ultracentrifugation for 20 min at 95,834 × g. The supernatant was loaded to a 5 ml Strep column (GE Healthcare) equilibrated with buffer A: 50 mM HEPES pH = 7.6, 200 mM sodium acetate, 0.1% digitonin, 0.05 mM TDAO. After extensive washing with buffer A, samples were eluted with buffer A + 2 mM desthiobiotin and 50 mM imidazole pH = 7.6 directly into 2 × 1 ml HisTrap columns (GE Healthcare) equilibrated with the same buffer. After additional washing step with buffer A containing 100 mM imidazole pH = 7.6, the protein complex was eluted with buffer A containing 350 mM imidazole pH = 7.6.

Sample of the T4SS_3‐10+D4 complex used for the NS‐EM experiments was in addition to the protocol mentioned above stabilized by mild cross‐link with 0.1% of glutaraldehyde (Sigma‐Aldrich) while being applied to the 10–30% sucrose density gradient at 50,512 × g for 15 h at 4°C using a SW40Ti rotor (Stark, 2010). Samples from the gradient were fractionated and analysed by SDS–PAGE. Fractions with bands at high molecular weight on the gels were analysed by NS‐EM. Grids of the samples which looked the most concentrated and least aggregated on the NS‐EM were used for collection of the images which were used for EM data processing.