The bacterial flagellum is a macromolecular protein complex that enables motility in many species. Bacterial flagella self-assemble a strong, multicomponent drive shaft that couples rotation in the inner membrane to the micrometre-long flagellar filament that powers bacterial swimming in viscous fluids1,2,3. Here, we present structures of the intact Salmonella flagellar basal body4, encompassing the inner membrane rotor, drive shaft and outer-membrane bushing, solved using cryo-electron microscopy to resolutions of 2.2–3.7 Å. The structures reveal molecular details of how 173 protein molecules of 13 different types assemble into a complex spanning two membranes and a cell wall. The helical drive shaft at one end is intricately interwoven with the rotor component with both the export gate complex and the proximal rod forming interactions with the MS-ring. At the other end, the drive shaft distal rod passes through the LP-ring bushing complex, which functions as a molecular bearing anchored in the outer membrane through interactions with the lipopolysaccharide. The in situ structure of a protein complex capping the drive shaft provides molecular insights into the assembly process of this molecular machine.
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Cryo-EM volumes and atomic models have been deposited at the EMDB (accession codes: EMD-12183, EMD-12190, EMD-12192, EMD-12193, EMD-12195) and PDB (accession codes: 7BGL, 7BHQ, 7BIN, 7BJ2, 7BK0), respectively. Proteomics data were deposited in PRIDE as PXD025599. Raw data and related Python code are available in Supplementary Data 2.
All code used for cryo-EM data analysis, structure determination and refinement are publicly available. Python code is available in Supplementary Data 2.
Berg, H. C. The rotary motor of bacterial flagella. Annu. Rev. Biochem. 72, 19–54 (2003).
Erhardt, M., Namba, K. & Hughes, K. T. Bacterial nanomachines: the flagellum and type III injectisome. Cold Spring Harb. Perspect. Biol. 2, a000299 (2010).
Nakamura, S. & Minamino, T. Flagella-driven motility of bacteria. Biomolecules 9, 279 (2019).
Minamino, T. & Imada, K. The bacterial flagellar motor and its structural diversity. Trends Microbiol. 23, 267–274 (2015).
Deme, J. C. et al. Structures of the stator complex that drives rotation of the bacterial flagellum. Nat. Microbiol. 5, 1553–1564 (2020).
Santiveri, M. et al. Structure and function of stator units of the bacterial flagellar motor. Cell 183, 244–257 (2020).
Kuhlen, L. et al. Structure of the core of the type III secretion system export apparatus. Nat. Struct. Mol. Biol. 25, 583–590 (2018).
Ueno, T., Oosawa, K. & Aizawa, S. M ring, S ring and proximal rod of the flagellar basal body of Salmonella Typhimurium are composed of subunits of a single protein, FliF. J. Mol. Biol. 227, 672–677 (1992).
Johnson, S. et al. Symmetry mismatch in the MS-ring of the bacterial flagellar rotor explains the structural coordination of secretion and rotation. Nat. Microbiol. https://doi.org/10.1038/s41564-020-0703-3 (2020).
Homma, M., Kutsukake, K., Hasebe, M., Iino, T. & Macnab, R. M. FlgB, FlgC, FlgF and FlgG. A family of structurally related proteins in the flagellar basal body of Salmonella Typhimurium. J. Mol. Biol. 211, 465–477 (1990).
Muller, V., Jones, C. J., Kawagishi, I., Aizawa, S. & Macnab, R. M. Characterization of the fliE genes of Escherichia coli and Salmonella Typhimurium and identification of the FliE protein as a component of the flagellar hook-basal body complex. J. Bacteriol. 174, 2298–2304 (1992).
Saijo-Hamano, Y., Matsunami, H., Namba, K. & Imada, K. Architecture of the bacterial flagellar distal rod and hook of Salmonella. Biomolecules https://doi.org/10.3390/biom9070260 (2019).
DePamphilis, M. L. & Adler, J. Fine structure and isolation of the hook-basal body complex of flagella from Escherichia coli and Bacillus subtilis. J. Bacteriol. 105, 384–395 (1971).
Jones, C. J., Homma, M. & Macnab, R. M. L-, P-, and M-ring proteins of the flagellar basal body of Salmonella Typhimurium: gene sequences and deduced protein sequences. J. Bacteriol. 171, 3890–3900 (1989).
Stafford, G. P., Ogi, T. & Hughes, C. Binding and transcriptional activation of non-flagellar genes by the Escherichia coli flagellar master regulator FlhD2C2. Microbiology 151, 1779–1788 (2005).
Sowa, Y. et al. Direct observation of steps in rotation of the bacterial flagellar motor. Nature 437, 916–919 (2005).
Thomas, D. R., Francis, N. R., Xu, C. & DeRosier, D. J. The three-dimensional structure of the flagellar rotor from a clockwise-locked mutant of Salmonella enterica serovar Typhimurium. J. Bacteriol. 188, 7039–7048 (2006).
Thomas, D. R., Morgan, D. G. & DeRosier, D. J. Rotational symmetry of the C ring and a mechanism for the flagellar rotary motor. Proc. Natl Acad. Sci. USA 96, 10134–10139 (1999).
Young, H. S., Dang, H., Lai, Y., DeRosier, D. J. & Khan, S. Variable symmetry in Salmonella Typhimurium flagellar motors. Biophys. J. 84, 571–577 (2003).
Nord, A. L., Sowa, Y., Steel, B. C., Lo, C. J. & Berry, R. M. Speed of the bacterial flagellar motor near zero load depends on the number of stator units. Proc. Natl Acad. Sci. USA 114, 11603–11608 (2017).
Mora, T., Yu, H., Sowa, Y. & Wingreen, N. S. Steps in the bacterial flagellar motor. PLoS Comput. Biol. 5, e1000540 (2009).
Hu, J. H. et al. T3S injectisome needle complex structures in four distinct states reveal the basis of membrane coupling and assembly. Nat. Microbiol. 4, 2010–2019 (2019).
Chevance, F. F. V. et al. The mechanism of outer membrane penetration by the eubacterial flagellum and implications for spirochete evolution. Genes Dev. 21, 2326–2335 (2007).
Cohen, E. J., Ferreira, J. L., Ladinsky, M. S., Beeby, M. & Hughes, K. T. Nanoscale-length control of the flagellar driveshaft requires hitting the tethered outer membrane. Science 356, 197–200 (2017).
Cohen, E. J. & Hughes, K. T. Rod-to-hook transition for extracellular flagellum assembly is catalyzed by the L-ring-dependent rod scaffold removal. J. Bacteriol. 196, 2387–2395 (2014).
Butan, C., Lara-Tejero, M., Li, W. W., Liu, J. & Galan, J. E. High-resolution view of the type III secretion export apparatus in situ reveals membrane remodeling and a secretion pathway. Proc. Natl Acad. Sci. USA 116, 24786–24795 (2019).
Fukumura, T. et al. Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex. PLoS Biol. 15, e2002281 (2017).
Kuhlen, L. et al. The substrate specificity switch FlhB assembles onto the export gate to regulate type three secretion. Nat. Commun. 11, 1296 (2020).
Ohnishi, K., Ohto, Y., Aizawa, S., Macnab, R. M. & Iino, T. FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella Typhimurium. J. Bacteriol. 176, 2272–2281 (1994).
Al-Otaibi, N. S. et al. The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation. Nat. Commun. https://doi.org/10.1038/s41467-020-16981-4 (2020).
Yonekura, K. et al. The bacterial flagellar cap as the rotary promoter of flagellin self-assembly. Science 290, 2148–2152 (2000).
Erhardt, M. & Hughes, K. T. C-ring requirement in flagellar type III secretion is bypassed by FlhDC upregulation. Mol. Microbiol. 75, 376–393 (2010).
Takaya, A. et al. YdiV: a dual function protein that targets FlhDC for ClpXP-dependent degradation by promoting release of DNA-bound FlhDC complex. Mol. Microbiol. 83, 1268–1284 (2012).
Sato, Y., Takaya, A., Mouslim, C., Hughes, K. T. & Yamamoto, T. FliT selectively enhances proteolysis of FlhC subunit in FlhD4C2 complex by an ATP-dependent protease, ClpXP. J. Biol. Chem. 289, 33001–33011 (2014).
Singer, H. M., Erhardt, M. & Hughes, K. T. RflM functions as a transcriptional repressor in the autogenous control of the Salmonella flagellar master operon flhDC. J. Bacteriol. 195, 4274–4282 (2013).
Karlinsey, J. E. λ-Red genetic engineering in Salmonella enterica serovar Typhimurium. Methods Enzymol. 421, 199–209 (2007).
Togashi, F., Yamaguchi, S., Kihara, M., Aizawa, S. I. & Macnab, R. M. An extreme clockwise switch bias mutation in fliG of Salmonella Typhimurium and its suppression by slow-motile mutations in motA and motB. J. Bacteriol. 179, 2994–3003 (1997).
Asai, Y., Yakushi, T., Kawagishi, I. & Homma, M. Ion-coupling determinants of Na+-driven and H+-driven flagellar motors. J. Mol. Biol. 327, 453–463 (2003).
Francis, N. R., Sosinsky, G. E., Thomas, D. & DeRosier, D. J. Isolation, characterization and structure of bacterial flagellar motors containing the switch complex. J. Mol. Biol. 235, 1261–1270 (1994).
Aizawa, S. I. in The Bacterial Flagellum Vol. 1593 (eds T. Minamino & K. Namba) 87–96 (Humana Press, 2017).
de la Mora, J., Camarena, L. & Dreyfus, G. in The Bacterial Flagellum Vol. 1593 (eds T. Minamino & K. Namba) 273–283 (Humana Press, 2017).
Martin, T. G., Boland, A., Fitzpatrick, A. W. P. & Scheres, S. H. W. Graphene oxide grid preparation. Figshare https://figshare.com/articles/media/Graphene_Oxide_Grid_Preparation/3178669/1 (2016).
Caesar, J. et al. SIMPLE 3.0 stream single-particle cryo-EM analysis in real time. J. Struct. Biol. 212, 107635 (2020).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife https://doi.org/10.7554/eLife.42166 (2018).
Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ 6, 5–17 (2019).
Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D. 71, 136–153 (2015).
Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 74, 531–544 (2018).
Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).
Ovchinnikov, S. et al. Large-scale determination of previously unsolved protein structures using evolutionary information. eLife 4, e09248 (2015).
Yuan, J. & Berg, H. C. Resurrection of the flagellar rotary motor near zero load. Proc. Natl Acad. Sci. USA 105, 1182–1185 (2008).
Sowa, Y., Steel, B. C. & Berry, R. M. A simple backscattering microscope for fast tracking of biological molecules. Rev. Sci. Instrum. 81, 113704 (2010).
Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).
Fitzgibbon, A., Pilu, M. & Fisher, R. B. Direct least square fitting of ellipses. IEEE Trans. Pattern Anal. Mach. Intell. 21, 476–480 (1999).
We thank E. Johnson and A. Costin of the Central Oxford Structural Molecular Imaging Centre (COSMIC) for assistance with data collection; H. Elmlund (Monash) for access to SIMPLE code ahead of release; N. Moriarty, P. Emsley and G. Murshudov for help with modelling lipid ligands. We acknowledge the use of the University of St Andrews BSRC Mass Spectrometry facility, and the Advanced Proteomics Facility of the Department of Biochemistry, University of Oxford, including M. Fournier for data processing. The Central Oxford Structural Molecular Imaging Centre is supported by the Wellcome Trust (no. 201536), The EPA Cephalosporin Trust, The Wolfson Foundation and a Royal Society/Wolfson Foundation Laboratory Refurbishment Grant (no. WL160052). Research in S.M.L.’s laboratory is supported by the Wellcome Trust Investigator (no. 219477) and Collaborative awards (no. 209194) and an MRC Programme Grant (no. S021264). A.L.N. is a member of the CBS, which is part of the France-BioImaging (FBI) and the French Infrastructure for Integrated Structural Biology (FRISBI), two national infrastructures supported by the French National Research Agency (ANR-10-INBS-04-01 and ANR-10-INBS-05, respectively).
The authors declare no competing interests.
Peer review information Nature Microbiology thanks Gert Bange, Marc Erhardt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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a, Example micrograph of Salmonella enterica serovar Typhimurium basal bodies on a graphene oxide surface. Scale bar 200 Å. b, 2D class averages of basal bodies. Scale bar 100 Å. c, Simplified workflow to show key stages in generation of each volume.
a, Gold-standard Fourier shell correlation (FSC) curve of RELION-postprocessed LP-ring C26 map. Resolution at the gold-standard cut-off (FSC = 0.143) is indicated. Curves: red, phase-randomized; green, unmasked; blue, masked; black, corrected. b, Local resolution estimates (in Å, as calculated by RELION3.1 LocalRes. c, Representative modelled densities of the three protein chains. d, Representative modelled densities of the lipid moieties.
a, Backscattering dark field setup. (M: mirror; L: lens; HWP: half wave plate; QWP: quarter wave plot; HeNe laser; fast CMOS camera). b, Schematic of the experimental assay: a gold nanoparticle of 100 nm is attached to the hook of a BFM on a surface-immobilised cell. c, Brightfield (left) and darkfield (right) image of a nanoparticle attached to the hook of a bacterial cell (scale bar is 1um). d-f, An example recording showing the 2D histogram of the position of the rotating nanoparticle, with 26-fold periodicity in rotation (d); the angular speed of the motor (median filtered, 20 ms window) (e); the kernel density distribution of the angular position of the motor (f). g, The average +/- S.D. (line and shading, respectively) of the weighted power spectrum of a histogram of the angular position. (27 motors recorded for 1–2 s, each from a different cell, where the sodium motive force ranged from 54–187 mV and speeds ranged from 2–300 Hz.). See Methods for details.
a, Gold-standard Fourier shell correlation (FSC) curve of RELION-postprocessed rod C1 map. Resolution at the gold-standard cutoff (FSC = 0.143) is indicated. Curves: red, phase-randomized; green, unmasked; blue, masked; black, corrected. b, Local resolution estimates (in Å), as calculated by RELION3.1 LocalRes. c, Representative modelled protein densities. d, Interaction of FliE (green cartoon and surface) with FliP of the Export Gate (blue cartoon). e, Overlay of a full FliE subunit (FliE-b) with FliE-a, demonstrating the overlap with the position of the FliR N-terminus. f, Overlay of representative rod subunits demonstrates the conserved core and variation in inserted domains (subunits colored as in Fig. 2).
a, Representation of the helical symmetry of the rod, with the different X-start helices highlighted. Protein subunits are represented as ellipses, colored as in Fig. 2. The lattice node shared by FliR and FliE-a is highlighted. b, Alternative representation of the lattice with ‘ghost’ additional protofilament to show how the structure closes.
a, Slab through the LP-rod demonstrating the thin seal around the rod. Green boxes highlight the constriction point formed by residues 48–82 of FlgI. b, Electrostatic potential mapped onto the surface of the LP-ring (view from inside the ring) with the positively charged band at the seal point highlighted in a green box). c, Electrostatic potential mapped onto the surface of the rod. Electrostatics analysed using APBS within Pymol.
a, Gold-standard Fourier shell correlation (FSC) curves of RELION-postprocessed P-ring C26 map. Resolution at the gold-standard cut-off (FSC = 0.143) is indicated. Curves: red, phase-randomized; green, unmasked; blue, masked; black, corrected. b, Volume (silver mesh) contoured at 0.018 electrons/Angstrom over one copy of FlgI (shown as ball-and-stick representation) c, Side by side views of the cut-away P-ring structure next to the intact basal body, aligned on the collar of the MS-ring/rod. The white dashed line highlights the centre of the P-ring assembly.
a, Gold-standard Fourier shell correlation (FSC) curve of RELION-postprocessed rod C1 cap map. Resolution at the gold-standard cutoff (FSC = 0.143) is indicated. Curves: red, phase-randomized; green, unmasked; blue, masked; black, corrected. b, Local resolution estimates (in Å), as calculated by RELION3.1 LocalRes. c, Representative modelled protein densities. d, Two-dimensional residue plot of FlgD showing co-variance between residue pairs, as calculated by Gremlin. Stronger co-variance is highlighted by darker colours. The N-terminal hairpin region is highlighted with a box.
Supplementary Figures 1–3 and references, and the legend for Supplementary Video 1.
Proposed motions involved in the FlgD-cap-catalysed assembly mechanism. The cap is shown as a surface, coloured as defined in Fig. 4c. The video (and Fig. 4f) shows a side view of sequential cap movements orientated as in Fig. 4e with the bacterial membranes below.
The proteomic identifications based on extraction from gel as described in the Methods.
Zip file containing a Python directory with full source data and the Python script used to analyse data and generate the plots shown in Fig. 1g, Extended Data Fig. 3 and Supplementary Figs. 2 and 3.
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Johnson, S., Furlong, E.J., Deme, J.C. et al. Molecular structure of the intact bacterial flagellar basal body. Nat Microbiol 6, 712–721 (2021). https://doi.org/10.1038/s41564-021-00895-y
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