Using template picking in cryoSPARC, ~1,400,000 Dot/Icm type IV secretion system (T4SS) particles were selected. The processing steps done in cryoSPARC are on a blue background, and the processing steps done using RELION are on a tan background. After 2D and 3D classification, the best class of ~136,000 particles was chosen for further refinement with and without C13 symmetry, resulting in reconstruction of 3D maps at 3.8 and 3.4 Å, respectively. The particle stack used in the refinement job in cryoSPARC was exported into Relion for further processing, such as contrast transfer function (CTF)-refinement and focused refinement. The 3.4 Å 3D model with C13 symmetry was used as an initial model for 3D structure determination in Relion using auto-3D refinement with C13 symmetry, resulting in reconstruction of 3D map of 3.8 Å resolution. Focused 3D classification (without alignment) including CTF per-particle refinement and then with either C13 (outer membrane cap [OMC]) or C18 symmetry (periplasmic ring [PR]) imposed was used to determine higher resolution maps of the OMC disk (with 13-fold symmetry) and the PR (with 18-fold symmetry). The maps of the OMC disk and PR were further refined using focused 3D refinement (with local refinement), resulting in 3D maps of the OMC disk (13-fold symmetry) and the PR (18-fold symmetry) at 2.8 Å for each region.