Molecular determination by electron microscopy of the dynein-microtubule complex structure.
Molecular determination by electron microscopy of the dynein-microtubule complex structure.
复制标题
通过电子显微镜对动力蛋白-微管复合物结构进行分子测定。
DOI：
10.1016/j.jmb.2007.07.046
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发表时间：
2007
影响因子：
5.6
通讯作者：
Maeda,Yuichiro
中科院分区：
文献类型：
--
作者：
Narita,Akihiro;Mizuno,Naoko;Kikkawa,Masahide;Maeda,Yuichiro
动力蛋白是一种负末端定向微管（MT）马达，负责真核细胞中广泛的基于MT的运动。动力蛋白化学机械转化的详细机制仍然是未知的，部分原因是动力蛋白的结构没有在高分辨率下研究。为了解决这个问题，并在更高的分辨率下重建动力蛋白-MT复合物，我们开发了基于单粒子分析的新程序。为了准确地确定动力蛋白-MT复合物的方向，我们引入了“动力蛋白轨道模型”来限制图像上可能的动力蛋白位置。我们测试了我们的程序，从模拟动力蛋白-MT复杂的图像重建结构。从使用三种不同模型的动力蛋白-MT复合物产生的模拟噪声图像开始，我们已经成功地恢复了原始的三维（3-D）结构。我们还表明，我们的程序是强大的动力蛋白分子的波动，即使在动力蛋白的位置波动到一定程度，可以确定的结构。最终的三维结构的收敛性可以用“二维（2-D）一致值”来测试，我们引入该值是为了查看最终的结构是否是波动的动力蛋白过拟合的结果。当程序由于波动而不能很好地工作时，我们可以通过该二维一致性值来识别故障。最后，从网骨藻细胞质动力蛋白-MT复合物的实际冷冻电子显微照片确定动力蛋白-MT复合物的实际结构。该方法揭示了动力蛋白-MT复合物的详细三维结构，并将有助于阐明动力蛋白分子的运动机制。
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.