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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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.
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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