Tomography without tilt: three-dimensional imaging of microtubule/motor complexes.
Usually structures such as microtubules are supposed to have surface lattices built from families of continuous helices, giving electron micrographs that can be analyzed by helical diffraction theory with a view to obtaining three-dimensional reconstructions. In the case of microtubules the helical surface lattice may be interrupted by discontinuities, called seams, in which case the usual helical reconstruction approach is no longer applicable. Even so, by virtue of their "superhelical" protofilaments, microtubules are still helical structures and we use this feature to treat a microtubule image as a set of projections equivalent to images obtained in a single axis tilt series. The main thrust of this article is to discuss how to obtain images and image parameters best suited to a tomographic approach to three-dimensional reconstruction. The method is tested by comparing helical and back-projection reconstructions of appropriate microtubules both with and without surface lattice decoration by kinesin family motor proteins. Tomographic reconstruction gives an independent demonstration that in vitro assembled microtubules have a B-type surface lattice. We show that 3-start, 15-protofilament microtubules have a seam, whereas 4-start microtubules have no seam and possess complete helical symmetry. Monomer motor domains attach to the outer ridge of the protofilaments and extend along the protofilament toward the plus end.
Metoz
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F
Laboratoire de Microscopie Electronique Structurale, Institut de Biologie Structurale (CEA/CNRS), Grenoble, France.
Arnal
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I
Wade
R H
RH
D000818:Animals; D004330:Drosophila; D016547:Kinesins / Q000737:chemistry; D008870:Microtubules / Q000648:ultrastructure*; D008958:Models, Molecular; D014054:Tomography; D014404:Tubulin / Q000737:chemistry
D014404:Tubulin; D016547:Kinesins