(a) Location of residues I167 and D253, which were predicted by the web server Disulfide by Design 2.0 (Craig and Dombkowski, 2013) to form stable disulfide bridges after mutation to cysteines. (b) FliD1–474(I167C/D253C) analyzed under reducing (lane 1) and non-reducing (lane 2) conditions by SDS-PAGE. (c) SAXS analysis of FliD1–474(I167C/D253C). Kratky plot (I*q2 versus q) and radial distribution function calculated by GNOM for 9.75 mg/mL (blue, used to calculate envelope), 4.88 mg/mL (red) and 2.44 mg/mL (grey). SAXS envelope calculated by DAMMIF with superimposed FliD78–405 crystal structure. (d) Swimming motility assay of wildtype PAO1 (WT), FliD transposon strain PW2975 (ΔfliD), ΔfliD complemented with FliD1–474 (ΔfliD/fliD1–474) or FliD1–474(I167C/D253C) (ΔfliD/fliD1–474(I167C/D253C)), respectively. (e) Western blot using anti-FliD scFv-Fc SH1579-B7 showing purified protein FliD1–474(I167C/D253C) under reducing (lane 1) and under non-reducing (lane 2) conditions. The presence of FliD in flagella preparations from wildtype PAO1 (lane 4), ΔfliD (lane 5), ΔfliD/fliD1–474(lane 6) and ΔfliD/fliD1–474(I167C/D253C) (lane 7) was analyzed under non-reducing conditions. The molecular weight standard is shown in lane 3 and the corresponding molecular weights are indicated on the right side of the blot. The 50 kDa and the 300 kDa bands representing FliD1–474 or hexameric FliD1–474(I167C/D253C), respectively, are indicated by red arrows.