Extended Data Figure 5: Analysis of interactions between subunit terminal helices within the crystallized mature hook (in support of Fig. 1).

a, Subunits crystallized in a lattice as they would be in the flagellum, with the terminal helices that line the channel forming intra-subunit antiparallel coiled-coils and inter-subunit lateral and axial packing interactions. Distances between engineered cysteine residues (Extended Data Table 1) were assessed between intra, axial and lateral subunits within the lattice, indicated by the open circles and dashed lines. This analysis allows the identification of crosslink residue pairs within subunit terminal helices that would form in an alternative coiled-coil conformation (parallel; Fig. 1d), distinct from subunits crystallized in the hook (antiparallel). b, Electron cryomicroscopy of the flagellar hook¹² (EMD 1647) carved to show four subunits within a lattice. Terminal helices are coloured as in a. c, Atomic model¹² of four subunits, coloured as in a, assembled as they would in the flagellar hook (PDB 3A69), derived from high-resolution electron cryomicroscopy and the atomic resolution structure of the hook subunit²⁴ (PDB 2BGZ). Subunit N-terminal helices and the C-terminal helix (light blue) of the upper central subunit are indicated by a dashed box. d, A magnification of c, showing N-terminal helices of four adjacent subunits (coloured as in a) and the C-terminal helix (light blue) of the upper central subunit (a). Distances (dashed lines) between specific selected engineered cysteine residues (yellow) are shown in Extended Data Table 1. e, Control crosslinking assay showing that FlgE-Ct without engineered cysteines, incubated with (+) or without (−) BMOE cross-linker (x-link), cannot form complexes with FlgE-Nt (no cysteine) or its mutated variants A₆C, A₁₄C, D₁₈C, A₂₅C or A₄₀C. All experiments were carried out at least three times and were biological replicates.