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. (C) 2015 Elsevier Inc. All rights reserved.