Abstract
Motility promotes the complex life cycle and infectious capabilities of Vibrio cholerae and is driven by rotation of a single polar flagellum. The flagellar filament comprises four flagellin proteins (FlaA–D) and is covered by a membranous sheath continuous with the outer membrane. Here we combine in situ cryo-electron microscopy single-particle analysis, fluorescence microscopy and molecular genetics to determine 2.92–3.43 Å structures of the sheathed flagellar filament from intact bacteria. Our data reveal the spatial arrangement of FlaA–D, showing that FlaA localizes at the cell pole and functions as a template for filament assembly involving multiple flagellins. Unlike unsheathed flagellar filaments, the sheathed filament from V. cholerae possesses a highly conserved core but a smooth, hydrophilic surface adjacent to the membranous sheath. A tiny conformational change at the single flagellin level results in a supercoiled filament and curved membranous sheath, supporting a model wherein the filament rotates separately from the sheath, enabling the distinct motility of V. cholerae.
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Data availability
The atomic coordinates and corresponding density maps of the flagellins have been deposited in the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB). The FlaA filament from the ΔflhG V. cholerae strain is available via PDB at 9N8G and EMD-49128. The FlaB filament from the same strain is available via PDB at 9P7R and EMD-71351. The FlaC filament from the ΔflhGΔflaBDE strain is available via PDB at 9N8M and EMD-49131. The FlaD filament from the ΔflhG strain is available via PDB at 9N8A and EMD-49125 (Supplementary Table 3).
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Acknowledgements
We thank J. Aronson for critical reading and editing of the paper. We thank C. Sindelar from Yale Center for Research Computing for providing support on cryo-EM data processing. W.G., J. Yue, J.W., J.G., J.M.B, R.K. and J.L. were partly supported by grants R01AI189907, R01AI087946 and R01AI132818 from the National Institute of Allergy and Infectious Diseases (NIAID) and National Institutes of Health (NIH). J.H.P. and F.H.Y. were supported by grants R01AI189907 and R01AI102584 from NIAID. V.S., H.H. and K.E.K. were supported by the Kleberg Foundation, Brown Foundation and San Antonio Area Foundation. J. Yan acknowledges the support from the National Science Foundation (MCB #2438891) and Simons Foundation International (SFI-LS-ECIAMEE-00006634). M.A. acknowledges the support from Charles H. Revson Foundation (25-19). J.-S.B.T. acknowledges the support from the Damon Runyon Cancer Research Foundation (DRG-2446-21). Cryo-EM data were collected at the Yale CryoEM Resource funded in part by NIH grant 1S10OD023603-01A1.
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Contributions
W.G. and J.L. conceived of the study. W.G. performed cryo-EM and cryo-ET data collection and processing. S.Z. initiated cryo-EM data processing of the non-sheathed flagellar filament structure. J.H.P. constructed the ΔflhG strain. V.S. and H.H. constructed the ΔflhGΔflaBDE and ΔflhGΔflaA strains for FlaC structural determination. M.A. and J.-S.B.T. performed flagellar labelling and fluorescence imaging of flagellins. J. Yue, J.W. and R.K. contributed to modelling, and J.G. contributed to manual picking of the flagellar tips and processing of the FlaB structure. J.M.B. produced the animation. S.W. assisted with data collection. J. Yan, K.E.K., F.H.Y. and J.L. provided critical resources, technical advice and supervision. W.G. and J.L. interpreted the data and wrote the paper with input from all authors. All authors discussed the results and approved the final version of the paper.
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Extended data
Extended Data Fig. 1 Multiple sequence alignment of flagellins FlaA-D.
Note that residues (259-267) are highlighted in blue. Residues (268-274, red) in FlaA exhibit a distinct morphology compared to other flagellins (FlaB, FlaC, and FlaD) due to two extra residues (K275 and T276, magenta).
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.
Extended Data Fig. 3 Cryo-EM maps at near-atomic resolution enable structure determination of four flagellins (FlaA, FlaB, FlaC, and FlaD) in V. cholerae.
(a-d) Representative model-to-map fits of FlaA, FlaB, FlaC, and FlaD into our cryo-EM maps derived from the in-situ cryo-EM map. Notably, the FlaD model fits well into the FlaD densities (left panels in a-d), while other models, including FlaA, FlaB, and FlaC, fit poorly into the FlaD densities (middle panels in a-d). The models of FlaA, FlaB, and FlaC fit well into their corresponding densities (right panels in a-d).
Extended Data Fig. 4 Comparison of flagellar filament structures across bacterial species.
(a) The flagellin model, filament model, surface electrostatic potential, and cryo-EM map from V. cholerae flagella are presented sequentially, left to right. The overall diameter of the sheathed filament is 270 Å. (b) The flagellin model, filament model, surface electrostatic potential, and cryo-EM map from S. Typhimurium15 flagella are presented sequentially, left to right. The overall diameter of the filament is 220 Å. (c) The flagellin model, filament model, surface electrostatic potential, and cryo-EM map from C. jejuni29 flagella are presented sequentially, left to right. The overall diameter of the filament is 180 Å. (d) The flagellin model, filament model, surface electrostatic potential, and cryo-EM map from P. aeruginosa27 flagella are presented sequentially, left to right. The overall diameter of the filament is 170 Å.
Extended Data Fig. 5 Interdomain interactions along the 11-start interface across bacterial species.
Three potential interaction pairs are: D940 -R5911, D3230 -R1611, and N3300 -M1211. Similar interactions are observed in other bacterial species, such as S. Typhimurium15 (E930 and G5911 or K5811, N4550 and L1111), P. aeruginosa27(T860 and N5211, T4340 and R1611, E4380 and L1211), and C. jejuni29 (T940 and N5911, T5220 and A1611, K5290 and L1211).
Extended Data Fig. 6 Interdomain interactions along the 5-start interface across bacterial species.
Our V. cholerae filament model presents three potential interaction pairs: E1220-R3145, K1360-D1525, and R660-D445. Similar interactions are observed in other bacterial species, such as S. Typhimurium15 (K1350 and N1505, E1210 and R4425, R650 and D425), P. aeruginosa27 (E1220 andR4255, and R660 and A465), and C. jejuni29 (E1220 and Q5135, and Q1360 and S1525).
Extended Data Fig. 8 CryoSPARC workflow for structural determination of the FlaA, FlaB, and FlaC filament in V. cholerae.
(a) 15,919 micrographs of ΔflhG V. cholerae were collected to determine the FlaA filament structure. Scale bar: 100 nm. (b) Representative 2D classifications of the hook–filament junction region after manual picking and Topaz picker30. Scale bar: 200 Å. (c, d) Cryo-EM density map of the hook–filament junction with FlaA filament, resolved at 3.43 Å based on the “gold standard” FSC0.143 plot. (e) Snapshot of manual picking at the filament tip region. Scale bar: 100 nm. (f) Representative 2D classifications of filament tip particles from 19,040 manually picked particles. Scale bar: 200 Å. (g, h) Cryo-EM density map of the FlaB filament at the filament tip, resolved at 3.16 Å. (i) Snapshot of filament tracer in ΔflhGΔflaBDE V. cholerae. Scale bar: 100 nm. (j) Representative 2D classifications of the FlaC filament. Scale bar: 200 Å. (k, l) Cryo-EM density map of the FlaC filament in ΔflhGΔflaBDE V. cholerae, resolved at 3.16 Å.
Extended Data Fig. 9 Cryo-ET reconstructions of the ΔflhGΔflaA cell poles.
(a) A slide shows a 55 nm flagellar hook. (b) A slide shows a short flagellum 100 nm in length.
Extended Data Fig. 10 The three-stranded β-sheet and helix motif on the filament surface are conserved across four flagellins in V. cholerae.
Four flagellins share similar morphology (top panels) and charged surfaces (bottom panels).
Supplementary information
Supplementary Information (download PDF )
Supplementary Tables 1–5.
Supplementary Video 1 (download MOV )
Animation of the FlaD filament structure.
Supplementary Video 2 (download MOV )
Animation of the FlaA filament structure.
Supplementary Video 3 (download MOV )
Animation of the FlaB filament structure.
Supplementary Video 4 (download MOV )
Animation of the whole filament in Vibrio cholerae.
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Guo, W., Zhang, S., Park, J.H. et al. Structures of the sheathed flagellum reveal mechanisms of assembly and rotation in Vibrio cholerae. Nat Microbiol 10, 3305–3314 (2025). https://doi.org/10.1038/s41564-025-02161-x
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DOI: https://doi.org/10.1038/s41564-025-02161-x
