title: DOLORS: versatile strategy for internal labeling and domain localization in electron microscopy.
abstract: Single-particle electron microscopy (EM) is a powerful tool for studying the structures of large biological molecules. However, the achievable resolution does not always allow for direct recognition of individual protein domains. Labels that can be visualized by EM have been developed for protein termini, but tagging internal domains remains a challenge. We describe a robust strategy for determining the position of internal sites within EM maps, termed domain localization by RCT sampling (DOLORS). DOLORS uses monovalent streptavidin added posttranslationally to tagged sites in the target protein. Internal labels generally display less conformational flexibility than terminal labels, providing more precise positional information. Automated methods are used to rapidly generate assemblies of unique 3D models allowing the attachment sites of labeled domains to be accurately identified and thus provide an overall architectural map of the molecule.
authors.affiliation: National Resource for Automated Molecular Microscopy, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Pick-Wei
authors.initials: PW
authors.lastname: Lau
authors.forename: Clinton S
authors.initials: CS
authors.lastname: Potter
authors.forename: Bridget
authors.initials: B
authors.lastname: Carragher
authors.forename: Ian J
authors.initials: IJ
authors.lastname: MacRae
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000648:ultrastructure; D029968:Escherichia coli Proteins / Q000737:chemistry; D006801:Humans; D008854:Microscopy, Electron / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000648:ultrastructure; D012097:Repressor Proteins / Q000737:chemistry; D043244:Ribonuclease III / Q000737:chemistry / Q000648:ultrastructure; D061987:Sf9 Cells; D013194:Staining and Labeling; D019809:Streptavidin / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D019809:Streptavidin; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli