J. Synchrotron Rad. (2011). 18, 2-5
Three-dimensional structural analysis of eukaryotic flagella/cilia by electron cryo-tomography
K. H. Bui , G. Pigino and T. Ishikawa
Figure 1 Structure of a flagellum and its components in various dimensions. The appropriate method for structural analysis at each scale is shown on the right. (a) Chlamydomonas cell with two flagella (5–10 µm length, 0.25 µm diameter). (b) Cross section [at the red dotted circle in (a)] of a flagellum. ODA: outer dynein arms. IDA: inner dynein arms. RS: radial spokes. (c) One microtubule doublet is extracted [red dotted circle in (b)], rotated and enlarged. (d) Schematic diagram of one dynein heavy chain [enclosed by the red dotted line in (c)]. (e) Atomic structure of the microtubule binding domain at the tip of the coiled-coil stalk.
Abstract
Electron cryo-tomography is a potential approach to analyzing the three-dimensional conformation of frozen hydrated biological macromolecules using electron microscopy. Since projections of each individual object illuminated from different orientations are merged, electron tomography is capable of structural analysis of such heterogeneous environments as in vivo or with polymorphism, although radiation damage and the missing wedge are severe problems. Here, recent results on the structure of eukaryotic flagella, which is an ATP-driven bending organelle, from green algae Chlamydomonas are presented. Tomographic analysis reveals asymmetric molecular arrangements, especially that of the dynein motor proteins, in flagella, giving insight into the mechanism of planar asymmetric bending motion. Methodological challenges to obtaining higher -resolution structures from this technique are also discussed.
Keywords: dynein ; flagella; axoneme ; tomography; cryo-EM.
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