Structural Analysis of FliI Ring Complexes

Purified proteins (final concentration 1 μM) was incubated in 50 mM Tris–HCl, pH 8.0, 113 mM NaCl, 0.8 mM EDTA, 1 mM DTT, 5 mM MgCl₂, 5 mM ADP, 5 mM AlCl₃, and 15 mM NaF at room temperature for 20 min. Samples were applied to carbon-coated copper grids and negatively stained with 2% (w/v) uranyl acetate. Electron micrographs were recorded at a magnification of ×50,000 with a JEM-1011 transmission electron microscope (JEOL, Tokyo, Japan) operated at 100 kV. To carry out two-dimensional class averaging of the FliI-3A, FliI-4A, and FliI-5A ring structures, 154, 100, and 103 particle images were picked manually, aligned, classified, and averaged using the RELION3.0.7 program^{45 (link)}.