期刊
JOURNAL OF STRUCTURAL BIOLOGY
卷 181, 期 2, 页码 169-178出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2012.11.004
关键词
Cryo-electron microscopy; Single-axis cryo-electron tomography; Dual-axis cryo-electron tomography; 3D Fourier transforms; Radiation damage; Microtubules
资金
- French National Agency for Research [ANR PCV06_142769, PCV07_190830]
- University of Rennes 1 [2011-12]
- Federative Research Institute Biosit [2012]
- Association for Cancer Research [2011-13]
- French Ministry of Education and Research
Single-axis cryo-electron tomography of vitrified specimens has become a method of choice to reconstruct in three dimensions macromolecular assemblies in their cellular context or prepared from purified components. Here, we asked how a dual-axis acquisition scheme would improve three-dimensional reconstructions of microtubules assembled in vitro. We show that in single-axis tomograms, microtubules oriented close to the perpendicular of the tilt axis display diminished contrast, and ultimately transform into sets of parallel lines oriented in the direction of the electron beam when observed in cross-section. Analysis of their three-dimensional Fourier transform indicates that this imaging artifact is due to a decrease in the angular sampling of their equatorial components. Although the second orthogonal series does not fully complement the first one at the specimen level due to increased radiation damage, it still allows elongated features oriented in any directions to be correctly reconstructed, which might be essential for highly heterogeneous specimens such as cells. (C) 2012 Elsevier Inc. All rights reserved.
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