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Probing a cell-embedded megadalton protein complex by DNP-supported solid-state NMR

Nature Methods volume 12, pages 649652 (2015) | Download Citation

Abstract

Studying biomolecules at atomic resolution in their native environment is the ultimate aim of structural biology. We investigated the bacterial type IV secretion system core complex (T4SScc) by cellular dynamic nuclear polarization–based solid-state nuclear magnetic resonance spectroscopy to validate a structural model previously generated by combining in vitro and in silico data. Our results indicate that T4SScc is well folded in the cellular setting, revealing protein regions that had been elusive when studied in vitro.

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Acknowledgements

We thank E. Koers and J. van der Zwan for helpful discussions and technical support. We are indebted to P. Tordo and his group for providing AMUPol. This work was funded in part by the Netherlands Organization for Scientific Research (NWO, grants 700.26.121 and 700.10.443 to M.B.)

Author information

Author notes

    • Abhishek Cukkemane

    Current address: Microbiology Department, Tuljaram Chaturchand College, Baramati, India.

Affiliations

  1. NMR Spectroscopy, Utrecht University, Utrecht, the Netherlands.

    • Mohammed Kaplan
    • , Abhishek Cukkemane
    • , Gydo C P van Zundert
    • , Siddarth Narasimhan
    • , Mark Daniëls
    • , Deni Mance
    • , Alexandre M J J Bonvin
    • , Gert E Folkers
    •  & Marc Baldus
  2. Institute of Structural and Molecular Biology, University College London and Birkbeck, London, UK.

    • Gabriel Waksman
  3. Groupe à 5 ans, Biologie structurale de la secretion bacterienne, Unité mixte de recherche Centre National de la Recherche Scientifique–Institut Pasteur 3528, Institut Pasteur, Paris, France.

    • Rémi Fronzes

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Contributions

G.E.F. and M.B. designed the research. M.K., M.D., A.C. and S.N. produced samples, and M.K. and D.M. performed ssNMR experiments. M.K., G.C.P.v.Z. and A.M.J.J.B. docked atomic models into the electron microscopy density map. R.F. provided clones and conducted electron microscopy studies. G.W. and M.B. wrote the paper and all authors edited it.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Gert E Folkers or Marc Baldus.

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    Supplementary Text and Figures

    Supplementary Figures 1–12 and Supplementary Tables 1–8

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    Supplementary Data

    Atomic models of the T4SS core complex in the EM density and of the N-terminal transmembrane helix of B10. The atomic model of the outer membrane complex of T4SScc (PDB 3JQO) was fitted into the EM density (EMD-2232) as a rigid body. The predicted transmembrane helix in the N-terminal part of VirB10 was modeled by imposing a helical conformation on the amino acid sequence. For the non-crystalline N-terminal part of VirB9, the already docked atomic model (PDB 2YPW)14 in the EM density (EMD = 2,232) was used.

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DOI

https://doi.org/10.1038/nmeth.3406

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