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Membrane-protein structure determination by solid-state NMR spectroscopy of microcrystals

Nature Methods volume 9, pages 12121217 (2012) | Download Citation

Abstract

Membrane proteins are largely underrepresented among available atomic-resolution structures. The use of detergents in protein purification procedures hinders the formation of well-ordered crystals for X-ray crystallography and leads to slower molecular tumbling, impeding the application of solution-state NMR. Solid-state magic-angle spinning NMR spectroscopy is an emerging method for membrane-protein structural biology that can overcome these technical problems. Here we present the solid-state NMR structure of the transmembrane domain of the Yersinia enterocolitica adhesin A (YadA). The sample was derived from crystallization trials that yielded only poorly diffracting microcrystals. We solved the structure using a single, uniformly 13C- and 15N-labeled sample. In addition, solid-state NMR allowed us to acquire information on the flexibility and mobility of parts of the structure, which, in combination with evolutionary conservation information, presents new insights into the autotransport mechanism of YadA.

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Acknowledgements

This work was supported by contract research 'Forschungsprogramm Methoden für die Lebenswissenschaften' of the Baden-Württemberg Stiftung to M.H. and D.L.; additional funding was from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 261863 (Bio-NMR) to B.B. and W.T.F., the Deutsche Forschungsgemeinschaft (HA 5918/1-1 to M.H., SFB 766 to D.L. and SFB 740 to B.-J.v.R. and W.T.F.), and institutional funds of the Leibniz Society and the Max Planck Society. The authors thank H. Oschkinat and A. Lupas for continuing support and H. Schwarz for his electron microscopy work.

Author information

Author notes

    • Ludwig Krabben

    Present address: Institut für Immunologie und Molekularbiologie, Fachbereich Veterinärmedizin, Freie Universität Berlin, Berlin, Germany.

    • Shakeel Ahmad Shahid
    •  & Benjamin Bardiaux

    These authors contributed equally to this work.

Affiliations

  1. Leibniz-Institut für Molekulare Pharmakologie, Abteilung Strukturbiologie, Berlin, Germany.

    • Shakeel Ahmad Shahid
    • , Benjamin Bardiaux
    • , W Trent Franks
    • , Ludwig Krabben
    •  & Barth-Jan van Rossum
  2. Max-Planck-Institut für Entwicklungsbiologie, Abteilung Proteinevolution, Tübingen, Germany.

    • Shakeel Ahmad Shahid
    • , Michael Habeck
    •  & Dirk Linke

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Contributions

L.K. and D.L. conceived of the project; B.-J.v.R. and D.L. designed the experiments; S.A.S., B.-J.v.R., W.T.F, L.K. and D.L. performed the experiments; S.A.S., M.H. and B.B. performed the structure calculations; and S.A.S., B.-J.v.R., M.H, B.B. and D.L. interpreted the data and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Michael Habeck or Barth-Jan van Rossum or Dirk Linke.

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DOI

https://doi.org/10.1038/nmeth.2248

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