title: Novel structural labeling method using cryo-electron tomography and biotin-streptavidin system.
abstract: There are a number of large macromolecular complexes that play important roles in the cell, and identifying the positions of their components is a key step to understanding their structure and function. Several structural labeling methods have been applied to electron microscopy in order to locate a specific component within a macromolecular complex, but each method is associated with problems in specificity, occupancy, signal intensity or precision. Here, we report a novel method for identifying the 3D locations of proteins using biotin-streptavidin labeling and cryo-electron tomography. We labeled a biotinylation-tagged intermediate chain of an axonemal dynein by streptavidin within the Chlamydomonas axoneme and visualized the 3D positions of the labels using subtomogram averaging. Increase of the density attributed to the bound streptavidin was validated by Student's t-test. In conclusion, the combination of the biotin-streptavidin system and cryo-electron tomography is a powerful method to investigate the structure of large macromolecular complexes.
authors.affiliation: Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Oda
authors.affiliation: Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address: mkikkawa@m.u-tokyo.ac.jp.
authors.forename: Masahide
authors.initials: M
authors.lastname: Kikkawa
mesh_terms: D000595:Amino Acid Sequence; D054468:Axoneme / Q000737:chemistry / Q000648:ultrastructure*; D001710:Biotin / Q000096:biosynthesis / Q000737:chemistry*; D016825:Chlamydomonas reinhardtii / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D055032:Electron Microscope Tomography; D021621:Imaging, Three-Dimensional; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013194:Staining and Labeling / Q000379:methods; D019809:Streptavidin / Q000737:chemistry*
chemical_list: D010940:Plant Proteins; D001710:Biotin; D019809:Streptavidin
keywords: Biotin–streptavidin; Chlamydomonas reinhardtii; Cilia and flagella; Cryo-EM; Cryo-ET; Cryo-electron tomography; Dynein; IC2; ODA; OID linker; Structural labeling; cryo-electron microscopy; cryo-electron tomography; intermediate chain 2; outer dynein arm; outer–inner dynein linker