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Precise mapping of subunits in multiprotein complexes by a versatile electron microscopy label

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Abstract

Positional knowledge of subunits within multiprotein assemblies is crucial for understanding their function. The topological analysis of protein complexes by electron microscopy has undergone impressive development, but analysis of the exact positioning of single subunits has lagged behind. Here we have developed a clonable ∼80-residue tag that, upon attachment to a target protein, can recruit a structurally prominent electron microscopy label in vitro. This tag is readily visible on single particles and becomes exceptionally distinct after image processing and classification. Thus, our method is applicable for the exact topological mapping of subunits in macromolecular complexes.

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Figure 1: Design of the DID–Dyn2 electron microscopy label.
Figure 2: Labeling of the Seh1 subunit within the Nup84 complex with the DID–Dyn2 electron microscopy marker.
Figure 3: Aligned class averages of unlabeled and DID–Dyn2–labeled Nup84 complex.

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Acknowledgements

We thank P. Bork and C. Müller for providing the facilities for transmission electron microscopy at the EMBL Heidelberg and M. Lutzmann (Institute of Human Genetics) for creating the pET24d-NUP85–SEH1 and pPROEXHtb-GST-TEV-NUP145C–SEC13-T7-NUP120 expression plasmids. K.T. is a recipient of the Kekulé grant from Fonds der Chemischen Industrie. E.H. is a recipient of grants from the Deutsche Forschungsgemeinschaft (SFB 638/B2) and Fonds der Chemischen Industrie.

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Authors and Affiliations

  1. Biochemie-Zentrum der Universität Heidelberg, Heidelberg, Germany

    Dirk Flemming, Karsten Thierbach, Philipp Stelter & Ed Hurt

  2. The School of Biological Sciences, University of Edinburgh, Scotland, UK

    Bettina Böttcher

Authors
  1. Dirk Flemming
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  2. Karsten Thierbach
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  3. Philipp Stelter
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  4. Bettina Böttcher
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  5. Ed Hurt
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Contributions

D.F., P.S. and E.H. initiated the project; D.F., K.T. and P.S. designed and performed the experiments; B.B. contributed to electron microscopy image processing and discussion; E.H. supervised the project.

Corresponding author

Correspondence to Ed Hurt.

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The authors declare no competing financial interests.

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Flemming, D., Thierbach, K., Stelter, P. et al. Precise mapping of subunits in multiprotein complexes by a versatile electron microscopy label. Nat Struct Mol Biol 17, 775–778 (2010). https://doi.org/10.1038/nsmb.1811

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