Extended Data Fig. 2: CryoSPARC workflow for structural determination of the FlaD filament in V. cholerae.

(a) A total of 2,145 micrographs were collected using a Titan Krios and processed in CryoSPARC. (b) Snapshots of filament tracer during particle picking. The representative images were acquired independently at least three times. Scale bar: 100 nm. (c) Representative 2D classifications showing unsheathed, sheathed, and curved flagellar filaments of ΔflhG V. cholerae. Scale bar: 200 Å. (d) Workflow of 3D reconstruction in CryoSPARC. Ab initio reconstruction was used to generate initial models, followed by homogeneous, helical, and local refinements to determine the helical twist and rise parameters. (e) Cryo-EM density map of the unsheathed flagellar filament after helical refinement, resolved at 2.45 Å based on the “gold standard” 0.143 FSC plot. (f) Cryo-EM density map of a sheathed, straight flagellar filament determined by homogeneous and local refinements without helical refinement, resolved at 2.92 Å. (g) Cryo-EM density map of a sheathed, curved flagellar filament resolved at 3.61 Å through homogeneous and local refinements without helical refinement.