Precise mapping of subunits in multiprotein complexes by a versatile electron microscopy label

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 approximately 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.