title: Protein domain mapping by internal labeling and single particle electron microscopy.
abstract: In recent years, electron microscopy (EM) and single particle analysis have emerged as essential tools for investigating the architecture of large biological complexes. When high resolution is achievable, crystal structure docking and de-novo modeling allows for precise assignment of individual protein domain sequences. However, the achievable resolution may limit the ability to do so, especially when small or flexible complexes are under study. In such cases, protein labeling has emerged as an important complementary tool to characterize domain architecture and elucidate functional mechanistic details. All labeling strategies proposed to date are either focused on the identification of the position of protein termini or require multi-step labeling strategies, potentially interfering with the final labeling efficiency. Here we describe a strategy for determining the position of internal protein domains within EM maps using a recombinant one-step labeling approach named Efficient Mapping by Internal Labeling (EMIL). EMIL takes advantage of the close spatial proximity of the GFP's N- and C-termini to generate protein chimeras containing an internal GFP at desired locations along the main protein chain. We apply this method to characterize the subunit domain localization of the human Polycomb Repressive Complex 2.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, United States. Electronic address: ciferric@gene.com.
authors.forename: Claudio
authors.initials: C
authors.lastname: Ciferri
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, United States.
authors.forename: Gabriel C
authors.initials: GC
authors.lastname: Lander
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, United States; Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, United States; Howard Hughes Medical Institute, UC Berkeley, Berkeley, United States.
authors.forename: Eva
authors.initials: E
authors.lastname: Nogales
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D008854:Microscopy, Electron / Q000379:methods*; D008958:Models, Molecular; D063151:Polycomb Repressive Complex 2 / Q000737:chemistry / Q000648:ultrastructure*; D017434:Protein Structure, Tertiary; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D049452:Green Fluorescent Proteins; D063151:Polycomb Repressive Complex 2
keywords: Domain mapping; Electron microscopy; GFP; Protein labeling; Structure