Molecular determination by electron microscopy of the dynein-microtubule complex structure.
Dynein is a minus-end-directed microtubule (MT) motor that is responsible for the wide range of MT-based motility in eukaryotic cells. Detailed mechanism of the dynein chemomechanical conversion is still unknown, partly because the structure of dynein is not studied at high resolution. To address this problem and reconstruct the dynein-MT complex at higher resolution, we have developed new procedures based on single particle analysis. To accurately determine the orientation of the dynein-MT complex, we introduced a "dynein track model" to restrict the possible dynein positions on the images. We tested our procedures by reconstructing structures from simulated dynein-MT complex images. Starting from the simulated noisy images generated using three different models of the dynein-MT complex, we have successfully recovered the original three-dimensional (3-D) structure. We also showed that our procedure is robust against fluctuation of the dynein molecules and can determine the structure even when the dynein position fluctuates to a certain extent. Convergence of the final 3-D structure can be tested with a "two-dimensional (2-D) agreement value," which we introduced to see whether the final structure is a result of overfit from fluctuating dynein or not. When the procedures did not work well due to the fluctuation, we could recognize the failure by this 2-D agreement value. Finally, the actual structure of the dynein-MT complex was determined from actual cryoelectron micrographs of Dictyostelium cytoplasmic dynein-MT complex. This method has revealed the detailed 3-D structures of the dynein-MT complex and will shed light on the motor mechanism of the dynein molecule.
Narita
Akihiro
A
ERATO Actin Filament Dynamics Project, Japan Science and Technology Agency, c/o RIKEN, Sayo, Hyogo 679-5148, Japan. narita@springs8.or.jp
Mizuno
Naoko
N
Kikkawa
Masahide
M
Maéda
Yuichiro
Y
D000818:Animals; D020285:Cryoelectron Microscopy; D004023:Dictyostelium / Q000737:chemistry; D004398:Dyneins / Q000378:metabolism / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure*; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000378:metabolism / Q000648:ultrastructure*; D015800:Protozoan Proteins / Q000378:metabolism / Q000648:ultrastructure
D046912:Multiprotein Complexes; D015800:Protozoan Proteins; D004398:Dyneins