a SEC chromatograms of YSD1_22 (green), a β-hairpin deletion mutant (delta-βHP, red), an N-terminal deletion mutant (delta-N, blue) and the double mutant (delta-βHP/delta-N, orange). Representative of three experiments from one (delta-βHP, double mutant) or two (YSD1_22, delta-N) purifications. The UV absorbance at 280 nm is displayed in milli-absorbance units (mAU). The asterisks indicate the peaks selected for negative-stain EM. The mass of delta-βHP was estimated to 231.7 kDa ± 4.2% by multi-angle laser light scattering. Theoretical mass: monomer = 38.7 kDa, hexamer = 232.2 kDa. b Representative negative-stain EM images of YSD1_22 and delta-βHP at ×67,000 magnification (two imaging sessions). c Zoomed view of ring-like objects highlighted by white arrows in b. For comparison, an hexameric ring of YSD1_22 is represented from the cryo-EM reconstruction of the assembled tail filtered to a resolution of 20 Å. Scale bars are 50 nm for b and 5 nm for c. d, e Orthogonal views of the hexameric rings forming the tail tube. For one subunit, domains 1, 2 and 3 and the β-hairpin (β-HP) are shown in yellow, brown, cyan and orange, respectively. Residues involved in inter-ring contacts are shown in magenta. Other subunits are coloured in grey and only one of these is shown in the side view of the ring. f A cross-section of the tail tube is shown as a ribbon within a brown molecular surface. Bottom-left inset: same view omitting β-HP. Top-left inset: zoom of the inter-ring interactions. Black and red boxes indicate contact areas with predominantly hydrophobic and polar interactions, respectively. g–i Model of the tail assembly process: Upon nucleation by the initiator (tail tip, represented as a blue triangle, or tail chaperones complex including the tape measure protein, represented as a blue cylinder), the N-terminus and β-HP form a splayed conformation (g), which is compatible with a stable hexameric ring (h). This conformation also positions the N-terminal arm and β-HP on either side of the rings making them available for axial polymerisation of the helical tail (i).