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J Struct Biol. 2013 Sep;183(3):305-11. doi: 10.1016/j.jsb.2013.07.003. Epub 2013 Jul 13.

Novel structural labeling method using cryo-electron tomography and biotin-streptavidin system.

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  • 1Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

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.

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

PMID:
23859837
DOI:
10.1016/j.jsb.2013.07.003
[PubMed - indexed for MEDLINE]
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