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J Struct Biol. 2015 Nov;192(2):151-8. doi: 10.1016/j.jsb.2015.09.010. Epub 2015 Sep 25.

Site-specific labeling of proteins for electron microscopy.

Author information

1
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.
2
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. Electronic address: glander@scripps.edu.

Abstract

Electron microscopy is commonly employed to determine the subunit organization of large macromolecular assemblies. However, the field lacks a robust molecular labeling methodology for unambiguous identification of constituent subunits. We present a strategy that exploits the unique properties of an unnatural amino acid in order to enable site-specific attachment of a single, readily identifiable protein label at any solvent-exposed position on the macromolecular surface. Using this method, we show clear labeling of a subunit within the 26S proteasome lid subcomplex that has not been amenable to labeling by traditional approaches.

KEYWORDS:

Electron microscopy; Molecular labeling; Unnatural amino acids

PMID:
26409249
PMCID:
PMC4633364
DOI:
10.1016/j.jsb.2015.09.010
[Indexed for MEDLINE]
Free PMC Article
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