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Structure of the hexagonal surface layer on Caulobacter crescentus cells

Nature Microbiology volume 2, Article number: 17059 (2017) Cite this article

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Abstract

Many prokaryotic cells are encapsulated by a surface layer (S-layer) consisting of repeating units of S-layer proteins. S-layer proteins are a diverse class of molecules found in Gram-positive and Gram-negative bacteria and most archaea1–5. S-layers protect cells from the outside, provide mechanical stability and also play roles in pathogenicity. In situ structural information about this highly abundant class of proteins is scarce, so atomic details of how S-layers are arranged on the surface of cells have remained elusive. Here, using purified Caulobacter crescentus' sole S-layer protein RsaA, we obtained a 2.7 Å X-ray structure that shows the hexameric S-layer lattice. We also solved a 7.4 Å structure of the S-layer through electron cryotomography and sub-tomogram averaging of cell stalks. The X-ray structure was docked unambiguously into the electron cryotomography map, resulting in a pseudo-atomic-level description of the in vivo S-layer, which agrees completely with the atomic X-ray lattice model. The cellular S-layer atomic structure shows that the S-layer is porous, with a largest gap dimension of 27 Å, and is stabilized by multiple Ca2+ ions bound near the interfaces. This study spans different spatial scales from atoms to cells by combining X-ray crystallography with electron cryotomography and sub-nanometre-resolution sub-tomogram averaging.

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Figure 1: Arrangement of the C. crescentus layer on cells and stalks.
Figure 2: The 2.7 Å X-ray structure of the outer S-layer lattice.
Figure 3: A 7.4 Å cryo-ET and sub-tomogram averaging map of the C. crescentus S-layer.
Figure 4: S-layer lattices observed in the X-ray structure and the cryo-ET map are exceptionally similar.

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Acknowledgements

The authors thank M. Skehel and F. Begum (MRC Laboratory of Molecular Biology, LMB) for mass-spectrometric identification of proteins, M. Yu (LMB) for help with X-ray data collection, C. Savva (LMB) for help with cryo-EM data collection, S. Scheres (LMB) for help with RELION software, F. Schur and W. Wan (European Molecular Biology Laboratory, EMBL) for providing high-resolution image-processing code and scripts before publication and for advice on their implementation, F. van den Ent (LMB) for advice on protein purification and T. Darling and J. Grimmett (LMB) for help with high-performance computing. The authors also thank Y. Modis (Cambridge University) for pointing out the similarity to anti-freeze proteins. Part of this work was funded by the European Molecular Biology Organization (aALTF 778-2015 to T.A.M.B.), the Medical Research Council (U105184326 to J.L.), the Wellcome Trust (095514/Z/11/Z to J.L.) and the National Institutes of Health (GM51986 to Y.V.B.). This work was supported by iNEXT, project no. 1482, funded by the Horizon 2020 programme of the European Union.

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Authors and Affiliations

  1. Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK

    Tanmay A. M. Bharat, Danguole Kureisaite-Ciziene, Ellen W. Yu, Jessica M. Devant, John A. G. Briggs & Jan Löwe

  2. Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK

    Tanmay A. M. Bharat

  3. Department of Biology, Indiana University, Bloomington, 47405, Indiana, USA

    Gail G. Hardy & Yves V. Brun

  4. Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, 69117, Germany

    Wim J. H. Hagen & John A. G. Briggs

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Contributions

T.A.M.B., Y.V.B. and J.L. designed the research. T.A.M.B., D.K.-C., G.G.H., E.W.Y., J.M.D., W.J.H.H. and J.L. performed experiments. W.J.H.H. and J.A.G.B. supported high-resolution cryo-ET and image processing. TA.M.B., D.K.-C., J.M.D., E.W.Y. and J.L. analysed the data. T.A.M.B. and J.L. wrote the manuscript with support from all authors.

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Correspondence to Tanmay A. M. Bharat or Jan Löwe.

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2; Supplementary Figures 1–7; legends for Supplementary Videos (PDF 2780 kb)

Supplementary Video 1

Whole cell cryo-ET of a C. crescentus cell (related to Figure 1). (AVI 21817 kb)

Supplementary Video 2

Cryo-ET of reconstituted 2D sheets of RsaA (related to Figure 2). (AVI 18787 kb)

Supplementary Video 3

The X-ray structure and map of a RsaA monomer (related to Figure 2). (MOV 35083 kb)

Supplementary Video 4

X-ray crystallography structure of the outer S-layer lattice (related to Figure 2). (MOV 35508 kb)

Supplementary Video 5

Cryo-ET of a C. crescentus stalk (related to Figure 3). (AVI 5543 kb)

Supplementary Video 6

Fit of the X-ray structure of the RsaA hexamer into the sub-tomogram averaging map (related to Figures 3,4). (AVI 28218 kb)

Supplementary Video 7

The structure and arrangement of the S-layer on cells (related to Figure 4). (AVI 46055 kb)

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Bharat, T., Kureisaite-Ciziene, D., Hardy, G. et al. Structure of the hexagonal surface layer on Caulobacter crescentus cells. Nat Microbiol 2, 17059 (2017). https://doi.org/10.1038/nmicrobiol.2017.59

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