期刊
JOURNAL OF STRUCTURAL BIOLOGY
卷 183, 期 3, 页码 305-311出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2013.07.003
关键词
Cryo-electron tomography; Chlamydomonas reinhardtii; Biotin-streptavidin; Cilia and flagella; Dynein; Structural labeling
资金
- Funding Program for Next Generation World-Leading Researchers [LS027]
- Japanese Ministry of Education, Culture, Sports Science and Technology
- Uehara Memorial Foundation
- Takeda Science Foundation
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. (c) 2013 Elsevier Inc. All rights reserved.
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