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Symmetry mismatch in the MS-ring of the bacterial flagellar rotor explains the structural coordination of secretion and rotation

Nature Microbiology volume 5, pages 966–975 (2020)Cite this article

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Abstract

The bacterial flagellum is a complex self-assembling nanomachine that confers motility to the cell. Despite great variation across species, all flagella are ultimately constructed from a helical propeller that is attached to a motor embedded in the inner membrane. The motor consists of a series of stator units surrounding a central rotor made up of two ring complexes, the MS-ring and the C-ring. Despite many studies, high-resolution structural information is still lacking for the MS-ring of the rotor, and proposed mismatches in stoichiometry between the two rings have long provided a source of confusion for the field. Here, we present structures of the Salmonella MS-ring, revealing a high level of variation in inter- and intrachain symmetry that provides a structural explanation for the ability of the MS-ring to function as a complex and elegant interface between the two main functions of the flagellum—protein secretion and rotation.

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Fig. 1: The overall structure of the flagellar MS-ring.
Fig. 2: Domain rearrangements required to build the full assembly.
Fig. 3: Conserved or co-varying residues map to the intersubunit interfaces.
Fig. 4: Comparison to structurally or functionally homologous assemblies.
Fig. 5: The flagellar MS-ring is structurally heterogenous.
Fig. 6: The MS-ring as a structural adaptor.

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Data availability

The data that support the findings of this study are available from the corresponding author on reasonable request. Cryo-EM maps have been deposited at the EMDB with the following accession codes: EMD-10143, EMD-10145, EMD-10146, EMD-10147, EMD-10148, EMD-10149, EMD-10560 and EMD-10561. Atomic coordinates have been deposited at the PDB with the following accession codes: 6SCN, 6SD1, 6SD2, 6SD3, 6SD4, 6SD5 and 6TRE.

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Acknowledgements

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; M. Beeby (Imperial College London) for access before publication to the P. shigelloides tomographic volume. The Central Oxford Structural Microscopy and Imaging Centre is supported by the Wellcome Trust (grant 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 Wellcome Trust Investigator (grant no. 100298) and Collaborative awards (no. 209194) and an MRC Programme Grant (no. MR/M011984/1). L.K. is a Wellcome Trust PhD student (no. 1009136).

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Author notes

  1. These authors contributed equally: Steven Johnson, Yu Hang Fong.

Authors and Affiliations

  1. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    Steven Johnson, Yu Hang Fong, Justin C. Deme, Emily J. Furlong, Lucas Kuhlen & Susan M. Lea

  2. Central Oxford Structural Molecular Imaging Centre, University of Oxford, Oxford, UK

    Justin C. Deme & Susan M. Lea

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Contributions

S.J. and S.M.L. designed the project, interpreted the data and wrote the first draft of the paper. S.J. analysed the data. Y.H.F. cloned, expressed and purified protein samples, and made and optimized EM grids. J.C.D. made and screened grids. J.C.D. and S.M.L. collected the EM data. E.J.F. expressed and purified samples and made EM grids. L.K. made constructs and performed preliminary purification experiments. All of the authors commented on drafts of the manuscript. Source data for Extended Data Fig. 3 are provided with the paper.

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Correspondence to Susan M. Lea.

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Extended data

Extended Data Fig. 1 Structure determination of 33mer.

a, SDS-PAGE of samples taken throughout purification of FliF in DDM. Lanes contain (1, 16) PageRuler Markers (2) whole cell lysate (3) supernatant from low speed spin (4) supernatant from high speed spin (5) solubilised membranes (6) supernatant from second low speed spin (7) supernatant from second high speed spin (8) resuspended pellet from second high speed spin (9–15 and 17–20) fractions from top to bottom of sucrose gradient post-high speed equilibration. Note—this gel shows samples from sucrose gradient without glutaraldehyde run in parallel with tubes containing glutaraldehyde. The fractions equivalent to those indicated (red box) were selected from the cross-linked gradients and used for structural analysis. b, Example micrograph (1.5 μm defocus) of cross-linked FliF on a graphene oxide surface. Scale bar 500 Å c, FSC curves from PostProcessing in RELIONv3.0 for volumes calculated in (i) C33, (ii) C21 and (iii) C3 respectively. d, Slab through C3 volume coloured by local resolution as estimated using RELIONv3.0.

Extended Data Fig. 2 33-fold symmetry does not resolve lower ring detail.

a, Volume generated by refinement in C33 shows lack of detail in RBM2inner region below, later explained by C21 symmetry in this region. b, Slab through central section of composite C33/C21/C3 volume reveals layered density derived from the detergent micelle at the periphery of the RBM3 ring and a central column of density below the C21 ring that presumably results from density associated with the 24 copies of RBM1 that are not located elsewhere in the map, the N-terminal trans-membrane helices attached to these and associated detergent.

Extended Data Fig. 3 Proteomic analysis reveals limited clipping at extreme C-terminus of FliF.

a, Purified, non-crosslinked S. Typhimurium FliF was run on blue native PAGE, then the gel band corresponding to the FliF complex was cut out and run on SDS-PAGE (lane marked X). The SDS-PAGE bands were cut out and mass spectrometry was used to identify the protein. The identity of each band is indicated on the gel. b, The three S Typhimurium FliF bands all produced peptides spread throughout the FliF amino acid sequence (that is from residues 2 to 560/552), however the two lighter bands had a lower intensity of peptides from the sequence post the folded RBM3 domain suggesting these bands differ in trimming of the extreme C-terminus beyond the structured region.

Source data

Extended Data Fig. 4 Comparisons of individual RBM domains.

a, Overlay of RBM2 and RBM3 domains of FliF (chain A), rmsd of 2.3 Å over 78 Cα. b, Superposition of the RBM2 domain of FliF on the closest structural homologue – the RBM2 of SctJ, rmsd of 1.1 Å over 79 Cα c. Superposition of the RBM3 domain of FliF on the SpoIIIAG RBM domain, rmsd of 2.3 Å over 80 Cα. The beta-insertions are not used to derive the superposition and the different relationship between the RBM domains and these inserts can therefore be appreciated. d, The N-terminal (blue) and C-terminal strands (red) of the beta-insert cross at the transition between tilted and vertically oriented strands.

Extended Data Fig. 5 Structural Observations.

a, The Electrostatic potential is mapped onto the surface of the FliF assembly (upper panel) and monomer using APBS within PyMol, revealing that the overall object is highly charged whilst the monomer interfaces are largely hydrophobic. b, A putative glutaraldehyde cross-link (marked with an asterisk) is observed in the beta-collar region of FliF.

Extended Data Fig. 6 Building the C21 portion of the structure.

a, Superposing a pair of neighbouring RBM3 domains on to a pair of neighbouring RBM2inner by aligning the first domain shows the rearrangements driven by the C33 versus C21 packing. b, The RBM2outer domains provide the major contact between the RBM2inner and RBM3 rings adapting between the C21 and C33 symmetries.

Extended Data Fig. 7 Subtle differences in RBM domain packing drives different assemblies.

a, Superposing a pair of neighbouring RBM3 domains on to a pair of neighbouring SpoIIIAG RBM domains by aligning the first domain shows the subtle alteration in packing needed to form the C33 rather than C30 assemblies. b, Superposing a pair of neighbouring RBM2inner domains on to a pair of neighbouring SctJ RBM2 domains by aligning the first domain shows the subtle alteration in packing needed to form the C21 rather than C24 assemblies.

Extended Data Fig. 8 Structure determination of 34mer.

a, FSC curves from PostProcessing in RELIONv3.0 for volumes calculated in (i) C34, (ii) C22 and (iii) C2 respectively. b, Slab through C2 volume coloured by local resolution as estimated using RELIONv3.0.

Extended Data Fig. 9 Supervised 3D classification reveals assembly diversity.

Distribution of particles between different symmetries in the RBM3 ring/β-collar region following supervised 3D-classification. 20% of particles were allocated to a C36 class, but the volume was uninterpretable from this class and presumably reflected damaged particles / particles with a variety of other symmetries.

Extended Data Fig. 10 Putative location of missing RBM1 domain in tomogram.

A slab through the centre of the P. shigelloides flagellar tomogram (grey surface; EMDB 10057) with the FliF C34 volume (blue surface) placed. The density of appropriate volume for the currently unresolved RBM1 domains is highlighted with red circles.

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Supplementary Data 1

Proteomic analysis of gel bands cropped from Extended Data Fig. 3.

Source data

Source Data Extended Data Fig. 3

Uncropped gel for Extended Data Fig. 3a.

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Johnson, S., Fong, Y.H., Deme, J.C. et al. Symmetry mismatch in the MS-ring of the bacterial flagellar rotor explains the structural coordination of secretion and rotation. Nat Microbiol 5, 966–975 (2020). https://doi.org/10.1038/s41564-020-0703-3

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