title: Site-specific labeling of proteins for electron microscopy.
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.
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Corey M
authors.initials: CM
authors.lastname: Dambacher
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. Electronic address: glander@scripps.edu.
authors.forename: Gabriel C
authors.initials: GC
authors.lastname: Lander
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D057930:Click Chemistry / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods; D046911:Macromolecular Substances / Q000032:analysis; D008854:Microscopy, Electron / Q000379:methods*; D008958:Models, Molecular; D046988:Proteasome Endopeptidase Complex / Q000032:analysis* / Q000737:chemistry*; D011506:Proteins / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000596:Amino Acids; D046911:Macromolecular Substances; D011506:Proteins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease
keywords: Electron microscopy; Molecular labeling; Unnatural amino acids