a, Amino-acid sequence of TssM. The different domains as well as the fragments used in this study are indicated (yellow, transmembrane helix; grey, cytoplasmic domain; green, blue and purple, periplasmic domain; blue and purple, C-terminal domain corresponding to the TssM32Ct fragment; purple, C-terminal domain corresponding to the TssM26Ct fragment). b, Crystal structure of the TssM32Ct–nb25 complex. The two proteins are represented as rainbow-coloured ribbons. The complementary determining regions (CDRs 1–3, coloured blue, green and red, respectively) of nb25 are indicated. The inset highlights the TssM32Ct–nb25 interface: the TssM32Ct surface is coloured beige whereas nb25 is represented as rainbow-coloured ribbons; the side chains of the amino acids in contact with TssM32Ct are indicated. The nb25 nanobody binds the TssM C-terminal domain, and covers a surface area of 580 Å2 by inserting its protruding CDR3 between TssM32Ct loops L5–6 and L9–10. The contacts between the two proteins are listed in Extended Data Table 2a. c, Crystal structure of the TssM26Ct–TssJ complex. Left: the two proteins are represented as ribbons and coloured in rainbow mode. Middle: same view rotated by 90°. The TssJ loop 1–2, previously shown to contact TssM25, is indicated. Right: TssM26Ct–TssJ interface. Top panel: the TssM26Ct surface is coloured violet, whereas TssJ is represented as rainbow-coloured ribbons. The TssJ side-chains of the amino acids in contact with TssM are indicated. The loops are numbered according to the flanking β-strands. Bottom panel: the TssJ surface is coloured beige whereas TssM26Ct is represented as rainbow-coloured ribbons. The TssM side-chains of the amino acids in contact with TssJ are indicated. The contacts between the two proteins are listed in Extended Data Table 2b. d, Comparison of the binding sites of nb25 and TssJ on TssM. Left: the structure of the TssM26Ct–TssJ complex (rainbow coloured) has been superimposed to the structure of the TssM32Ct–nb25 complex (only nb25 is shown in grey for clarity). Right: the same partners as in the left panel in surface representation. TssM26Ct (violet), TssJ (green) and nb25 (pink). e, Insertion of the TssJ lipid anchor in the outer membrane. Left: TssJ structure25 with the N-terminal 24 residues (absent in the crystal structure). This N-terminal extension (in magenta), predicted to be disordered, was modelled in Chimera using Modeller. The first cysteine residue is acylated to allow anchorage to the inner leaflet of the outer membrane (orange rectangle). Right: docking of the TssM26Ct–TssJ complex in the EM 3D reconstruction of the TssJLM complex (only the uppermost (tip) part of the TssJLM complex is shown). Left panel: two TssM26Ct–TssJ were docked into the inner and outer pillars of the tip complex. Right panel: docking in each pillar of the TssJLM tip complex (C5 symmetry). f, Hydrophobicity of the TssM26Ct–TssJ complex. Surface representation of the TssM26Ct–TssJ decamer (left, top view; right, side view). The hydrophobicity of the surface residues is displayed (blue to red scale from most hydrophilic to most hydrophobic). No obvious hydrophobic patch is visible at the surface of the complex.