Site-specific labeling of proteins for electron microscopy

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / chemistry
  • Click Chemistry / methods
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Imaging, Three-Dimensional / methods
  • Macromolecular Substances / analysis
  • Microscopy, Electron / methods*
  • Models, Molecular
  • Proteasome Endopeptidase Complex / analysis*
  • Proteasome Endopeptidase Complex / chemistry*
  • Proteins / analysis*
  • Staining and Labeling / methods*


  • Amino Acids
  • Macromolecular Substances
  • Proteins
  • Proteasome Endopeptidase Complex
  • ATP dependent 26S protease