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Structures of the stator complex that drives rotation of the bacterial flagellum

An Author Correction to this article was published on 09 November 2020

This article has been updated


The bacterial flagellum is the prototypical protein nanomachine and comprises a rotating helical propeller attached to a membrane-embedded motor complex. The motor consists of a central rotor surrounded by stator units that couple ion flow across the cytoplasmic membrane to generate torque. Here, we present the structures of the stator complexes from Clostridium sporogenes, Bacillus subtilis and Vibrio mimicus, allowing interpretation of the extensive body of data on stator mechanism. The structures reveal an unexpected asymmetric A5B2 subunit assembly where the five A subunits enclose the two B subunits. Comparison to structures of other ion-driven motors indicates that this A5B2 architecture is fundamental to bacterial systems that couple energy from ion flow to generate mechanical work at a distance and suggests that such events involve rotation in the motor structures.

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Fig. 1: Stator complexes from multiple organisms have a MotA5MotB2 stoichiometry.
Fig. 2: Structures of stator complexes from C. sporogenes and B. subtilis.
Fig. 3: Functionally critical regions of the stator complex.
Fig. 4: Mechanistic model for the generation of bidirectional flagellar torque.
Fig. 5: Conservation of core architecture between diverse families of ion-driven motors.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Cryo-EM volumes and atomic models have been deposited with the Electron Microscopy Data Bank (accession nos. EMD-10895, EMD-10899, EMD-10901, EMD-10902, EMD-10897) and Protein Data Bank (accession nos. 6YSF and 6YSL), respectively. Source data are provided with this paper.

Change history

  • 09 November 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.


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We thank D. Blair for providing E. coli RP6894. We thank E. Johnson and A. Costin of the Central Oxford Structural Molecular Imaging Centre (COSMIC) for assistance with data collection and H. Elmlund (Monash) for access to the SIMPLE3.0 code ahead of release. We acknowledge the use of COSMIC. COSMIC is supported by the Wellcome Trust (grant no. 201536), EPA Cephalosporin Trust, Wolfson Foundation and a Royal Society/Wolfson Foundation Laboratory Refurbishment Grant (no. WL160052). Research in S.M.L.’s laboratory is supported by a Wellcome Trust Investigator Award (grant no. 100298), a Collaborative Award (no. 209194) and a Medical Research Council (MRC) Programme Grant (no. MR/M011984/1). Research in B.C.B.’s laboratory is supported by a Wellcome Trust Investigator Award (grant no. 107929/Z/15/Z). Research in J.W.C.’s laboratory is supported by the Canadian Institutes of Health Research (grant no. 178048-BMA-CFAA-11449). Research in P.J.S.’s lab is funded by the Wellcome Trust (grant no. 208361/Z/17/Z), MRC (grant no. MR/S009213/1) and BBSRC (grant nos. BB/P01948X/1, BB/R002517/1 and BB/S003339/1). This project made use of time on ARCHER and JADE granted via the UK High-End Computing Consortium for Biomolecular Simulation (, supported by the Engineering and Physical Sciences Research Council (EPSRC) (grant no. EP/R029407/1) and Athena at HPC Midlands+, which was funded by the EPSRC (grant no. EP/P020232/1), and used the University of Warwick Scientific Computing Research Technology Platform for computational access.

Author information

Authors and Affiliations



J.C.D. carried out all biochemical work except as credited otherwise, prepared the cryo-EM grids, collected and processed the EM data and determined the structures. J.C.D., S.J. and S.M.L. designed the project, interpreted the data, built the models and wrote the first draft of the paper. S.J. performed the MALS experiments. O.V. and P.J.S. performed the molecular dynamics simulations. A.A., H.M. and T.G. carried out the biochemical work on Pseudomonas TonB-ExbB-ExbD. R.H.J. and B.C.B. contributed to the GldLM structure. J.W.C. initiated and provided materials for the ExbBD project. All authors commented on drafts of the manuscript.

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Correspondence to Susan M. Lea.

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Extended data

Extended Data Fig. 1 Ion specificity of stator complexes.

Sequence alignment of the (a) transmembrane and plug helices of MotB and (b) transmembrane helices (TMHs) 3 and 4 of MotA from E. coli and other relevant bacterial species. Species are classified based on ion specificity (red, H+-driven; blue, Na+-driven). Residues that are conserved just within either the H+ or Na+-driven classes of stator and map to the MotA-MotB interface (c) are indicated with orange asterisks in the alignment and same residue colouring across H+-driven (Left) and Na+-driven (Right) models (using E. coli MotAB numbering equivalents). The threonine ring is highlighted with light blue asterisks (a, b) or residues (c). Pairwise identity tables of MotB and MotA species used in alignments are found in Supplementary Tables 5 and 6, respectively. The ion used by C. sporogenes is ambiguous in that it is annotated as a proton-driven system, but the sequence alignment has more commonality with the sodium driven stators. We therefore do not colour it as definitively belonging to either class.

Extended Data Fig. 2 Cryo-EM map quality and resolution estimates of stator complexes.

a, Gold-standard Fourier shell correlation (FSC) curves of RELION-postprocessed stator complex volumes. 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) of the sharpened volumes. c, Representative modelled densities.

Extended Data Fig. 3 Conservation, covariance, and prior mutagenesis data mapped onto the stator complex structure.

a, Surface conservation as determined by Consurf51 (maroon high conservation, cyan low conservation). (Left) View from the cytoplasm showing conservation at the cytoplasmic MotA domains, including residues previously identified to be important for torque generation25 (R90 and E98). (Centre) Side view showing poor conservation within membrane-interfacing residues of MotA. (Right) Cutaway displaying high level of conservation at the MotA-MotB interface; MotA shown as surface representation, MotB as ribbon. bd, Evolutionary co-variation of residues (b) within MotA, (c) between MotA subunits, with boxes highlighting regions of strong covariance that are illustrated in more detail in the adjacent fragments, or (d) between MotA and MotB with the left hand side showing contacts for modelled MotB regions and the right hand side showing contacts in the unmodelled N-terminal MotB density represented here as a poly-alanine backbone. Predictions were carried out in Gremlin6 and contacts with a probability score of > 0.9 are shown. Contacts are coloured by Cα-Cα distance (≤10 Å in green, ≤15 Å in yellow). e, Mapping previous tryptophan scanning mutagenesis performed on MotA TMHs23 (i) or MotB24 (ii) to the stator complex structure. MotA and MotB are coloured as in Figs. 2 and 3 with targeted residues coloured according to toleration to mutagenesis; yellow corresponds to tolerated mutants (relative swarm rates > 0.5), red are poorly tolerated mutants (relative swarm rates of ≤ 0.5). In (i), TMH4 (green) and TMH2 (red) of neighbouring MotA subunits and MotB (white) are shown as transparent silhouettes. Poorly tolerated mutants cluster at subunit interfaces. In (ii) only TMH3-TMH4 of three MotA subunits are shown for clarity. f, Mapping previously determined cysteine crosslinks between (i) MotA-MotA21, (ii) MotB-MotB22, or (iii) MotA-MotB21 to our structure. For displaying crosslinks, a yield of ≥ 30% disulfide-linked adduct under iodine oxidizing conditions was used as threshold, except for MotATMH4-MotB crosslinks which used a ≥ 10% threshold. All analyses in this figure were performed using an E. coli MotAB structure generated by homology threading onto C. sporogenes MotAB.

Extended Data Fig. 4 Residues that interact with the flagellar C-ring form a charged ring on the cytoplasmic face of MotA.

a, An isolated MotA subunit with the essential torque-generating charged residues R90 and E9825 displayed in yellow spheres representation. b, The full C. sporogenes stator complex viewed from the side (Left) or from the cytoplasm (Right), coloured as in Figs. 2 and 3, and with the torque-generating charged residues represented as in (a) using E. coli MotAB numbering scheme.

Extended Data Fig. 5 Structural elements of the B. subtilis stator complex.

a, Structure of the B. subtilis stator complex depicting unmodeled density for the MotB N-terminal extensions (Left) and plug helices (Right). Inset shows plug densities in context of top of complex. b, (i) Isolated B. subtilis MotB dimer represented as in (a), and (ii) superposition of the TMHs of the two MotB chains showing the relative rotation of the plug helices. The location of the membrane is indicated in grey. c, Slabs at the heights indicated through B. subtilis MotAB show distortion from a regular pentagon (arrow indicates the cytoplasmic side of the complex) as viewed from the cytoplasm). d, Surface representation of B. subtilis MotAB showing tight packing. (Top) Side view with the front of the complex removed. (Bottom) Top-down view of the slab indicated by dashed lines. e, Structural alignment of the five B. subtilis MotA chains reveal they fall into two conformational classes which differ in the degree of flexing at the highlighted prolines.

Extended Data Fig. 6 Overview of differences in MotB environment caused by asymmetry of stator complex.

a, Each chain of MotB is shown with its respective surrounding MotA subunits, superposed by overlay of MotB30-34 (that is a turn of helix centered on the critical D32). MotB-chain A and its two neighbouring MotA subunits (chains E and D) shown in orange tones, MotB-chain C and its three neighbouring MotA subunits (chains C, F & G) shown in green b, same representation as a but rotated 90 degrees c & d show each MotB and neighbouring MotA chains, coloured as in a & b but with closest approaches between critical residue pairs shown to highlight the very different arrangement of the MotA subunits w.r.t. MotB D32. All panels drawn using PyMol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).

Extended Data Fig. 7 Molecular dynamics simulations of stator complex structures in lipid bilayers.

a, Side (top) and top-down (bottom) views of (i) C. sporogenes and (ii) B. subtilis MotAB inserted within a lipid bilayer after extended simulations (coarsegrain for 1 µs then atomistic for a further 200 ns). b, Cartoon representation of (i) C. sporogenes MotAB and (ii) B. subtilis MotAB coloured (blue to red) by the average rmsf of the 3 replica simulations performed.

Extended Data Fig. 8 Cryo-EM map quality and resolution estimates of ExbBD complexes.

a, Gold-standard Fourier shell correlation (FSC) curves of RELION-postprocessed ExbBD maps. 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) of the sharpened maps. c, Structure of the 5:2 ExbBD complex from E. coli37 (PDB 6tyi) fit into the E. coli W (top) and P. savastanoi ExbBD (bottom) maps. ExbB is coloured dark grey and ExbD is coloured blue. Top right panels have three ExbB subunits removed to demonstrate density for the two TMHs of ExbD. Bottom right panels reveal the 5:2 ExbB:ExbD arrangement as viewed from the cytoplasm.

Extended Data Fig. 9 Structural alignment of MotAB and ExbBD.

a, side views of the cores of the C. sporogenes MotAB (orange) and E. coli ExbBD37 (yellow) complexes with two front MotA/ExbB subunits removed showing the conserved Asp residues lying at the same height with respect to a ring of Thr on the MotA/ExbD components b, Overlaying the cores of C. sporogenes MotAB (orange) and E. coli ExbBD (yellow) by alignment of the MotA/ExbB helices demonstrates alignment of the critical polar residues between the two systems. Regions outside the core shown as transparent grey; MotB/ExbD shown in grey. c, Aligning a single subunit of MotA (rainbow colouring) with ExbB (white) via the pore lining helices reveals the different elaborations of this core unit in each protein. d, Aligning a single subunit of MotA with ExbB via the cytoplasmic extensions of the two core helices reveals the different folding of the rest of the cytoplasmic regions. Proteins coloured as in (c).

Extended Data Fig. 10 TonB recruitment to the ExbBD complex.

a, SEC-MALS profile of purified Pseudomonas TonB-ExbBD (black curve; absorbance at 280 nm (A280)) and ExbBD (grey curve; absorbance at 280 nm (A280)) with SDS-PAGE analysis of each sample inlayed. Total protein-detergent complex molar mass (green) and deconvoluted protein (blue) and detergent (orange) molar masses are shown. A ~30 kDa difference in molar mass is observed between the complexes consistent with the TonB-ExbBD complex containing one TonB subunit. Similar data were obtained from three independent purifications. b, Evolutionary co-variation of residues between TonB and ExbB displayed on the E. coli ExbBD coordinates37 (PDB 6tyi). For clarity, a topological model of the TMH of TonB is shown in orange with TonB-ExbB contacts indicated as dashed lines. ExbB residues that coevolve with TonB decorate the external surface of ExbB and are displayed in green. ExbB (grey) and ExbD (blue) are displayed as ribbon cartoons. Contacts shown were generated by Gremlin6 and have a probability score of > 0.9. c, inspection of the cryo-em volume derived from the ExbBDTonB sample, prior to post-processing, at a low contour level reveals a single, tube of density, consistent with the predicted location of the TonB transmembrane helix running across the exterior of a single copy of ExbB. Although likely at a low occupancy c.f. the other components, we tentatively assign this as TonB.

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Supplementary Data 1

Uncropped gels for Supplementary Fig. 11.

Supplementary Data 2

Uncropped gels for Supplementary Fig. 1.

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Uncropped gels for Extended Data Fig 10a.

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Deme, J.C., Johnson, S., Vickery, O. et al. Structures of the stator complex that drives rotation of the bacterial flagellum. Nat Microbiol 5, 1553–1564 (2020).

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