Extended Data Fig. 1. Prior knowledge of conjugative T4SS architectures and proteins, purification and cryo-EM analysis of the R388 T4SS.

a, Known 3D architectures of conjugative T4SSs. Left and middle: front and side views of the low resolution negative-stained EM structure of the R388 T4SS (EMD-2567)⁸. Right: low resolution cryo-electron tomography structure of the F T4SS (EMD-9344 and EMD-9347)¹⁰. The IMC, Stalk, Arches, and OMCC are colour-coded as in Fig. 1b. b, Primary structure of the Trw/VirB proteins observed in this study. Each protein is named TrwX/VirBX according to convention where the first name is that of the Trw protein in the trw R388 plasmid gene cluster, and the second name is the name of its homologue in the Agrobacterium system. Each protein is shown as a hashed rectangle, the length of which is proportional to the length of its sequence. Colour-coding for each protein is as in Fig. 1d. Transmembrane segments as observed in the structure are shown in boxes labelled “TM”. Signal sequences are shown in empty boxes labelled SP. Lines under the rectangles indicate the parts of the sequence for which the electron density was of high enough quality to build a complete atomic model including side chains (in green), or where the secondary structures definition was good enough to build main chain secondary structures but not side chains (in red), or was so poor that no model could be built (in black). Boundary residue numbering for each box and line are indicated. Table at right recapitulates, for each protein, the proportion of the sequence either built with side chains, or with built main chain only, or missing in our structure. c, SDS-PAGE analysis of the purified R388 T4SS (For gel source data, see Supplementary Fig. 1a and corresponding legend in SI guide; n = 9 independent experiments). Molecular weight markers are indicated on the left. The proteins bands (identified by mass spectrometry) are labelled on the right. * and ** indicate minor contaminants (OmpA and OmpC). We purified a T4SS complex as in Redzej et al. (2017)⁹ i.e. a complex composed of 9 of the essential Trw/VirB proteins, TrwM/VirB3-TrwE/VirB10 and TrwB/VirD4 (Extended Data Fig. 1c), except that 1- the His-tag column purification step to enrich the preparation with VirD4-bound complexes was not carried out, 2- the complex was not crosslinked, a step shown by Redzej et al. (2017)⁹ to be required to keep TrwB/VirD4 bound, 3- concentration of the complex was achieved using ultracentrifugation, and 4- potential aggregates resulting from concentration were separated using sucrose density gradient centrifugation (see Methods). TrwB/VirD4 was therefore absent from the structure as it needs crosslinking to remain bound⁹. Association of VirD4 with the T4SS may need stabilising through its interaction with the DNA substrate or its dissociation might be triggered by the cryogenic conditions. TrwD/VirB11 was also not part of the complex since it dissociates in the presence of detergents (Extended Data Fig. 1d). As a result, the structure contains TrwM/VirB3-TrwE/VirB10, a complex which was previously examined by negative stain EM (NSEM) by Low et al. (2014)⁸ and found to adopt the double-barrelled architecture. However, the purification protocol used to purify the complex here was greatly modified compared to that used in Low et al. (2014)⁸. Crucial to the improved conditions used here was the use of TDAO, a zwitterionic detergent. Moreover, concentration and separation from aggregates were also changed: in Low et al. (2014)⁸, the complex could not be concentrated without heavily aggregating, while here, due to a change in detergents mix, concentration using ultra-centrifugation was achieved as well as removing of minority aggregates by sucrose density gradient centrifugation. As a result of these significant modifications in the purification protocol, the yields were greatly improved (assessed to being between 20-30-fold), the complex being also much less prone to aggregation. Improved yields and higher quality sample combined to make the determination of this complex structure by cryo-EM possible. As explained in main text, a minority of particles in the cryo-EM data set presented here display the typical side views of the double-barrelled structure, indicating that the majority hexamer of dimers architecture we observe here must have been unstable in the buffer and NSEM conditions used by Low et al. (2014)⁸ since they did not observe it. In contrast, yields and stability of the double-barrelled complex obtained by Low et al. (2014)⁸ were low, making it impossible to solve its cryo-EM structure. d, SDS-PAGE analysis of the purified TraB/VirB4-TraG/VirB11 complex in the absence (No detergent) or presence of the detergents used to extract the T4SS (+detergent). For gel source data, see Supplementary Fig. 1b and corresponding legend in SI guide. n=3 independent experiments. e, Cryo-EM micrograph of the R388 T4SS. Red circles indicate examples of particles. 104,711 such micrographs over 7 datasets were collected. f, 2D classes found in the cryo-EM data set that show a view similar to that of the side views of the double-barrelled architecture observed by Low et al. (2014)⁸. The double-barrelled structure is characterised by a unique side view shown in Extended Data Fig. 1a, middle panel. Therefore, we asked whether such side views could be found in the cryo-EM data set described here. Left: an example of side view 2D classes (labelled “2D-class”) typically found in the NSEM double-barrelled architecture data and a corresponding 2D projection from the NSEM double-barrelled map (labelled “2D projection from 3D”). Right: 2 examples of similar side views but in the cryo-EM data set presented here. These 2D classes were generated using 2D classification of the set of 1,292,734 particles mentioned in Extended Data Fig. 1i, and selected for their resemblance to the side-view projections shown at left, resulting in the final selection of about 4,838 particles, i.e. circa 0.3% of the data set.