High-Resolution Structural Analysis of Dyneins by Cryo-electron Microscopy.
High-Resolution Structural Analysis of Dyneins by Cryo-electron Microscopy.
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DOI：
10.1007/978-1-0716-2958-1_16
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发表时间：
2023
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低温电子显微镜（cryo-EM）已成为研究大分子结构的主流技术。结构生物学家比以往任何时候都更容易确定蛋白质复合物的结构。尽管如此，获得动力蛋白等分子马达的高分辨率结构仍然是一个极具挑战性的目标，因为它们在冰中的麻烦行为、极其灵活的构象以及复杂的结构。动力蛋白是一个大的分子机器，驱动许多基本的细胞货物的运动，也是动力纤毛运动的关键力发生器。动力蛋白在不同状态下的高分辨率结构信息对于深入理解其在细胞中的作用机制至关重要。在这里，我们总结了冷冻电镜的方法，我们已经用来研究外臂动力蛋白阵列的结构绑定到微管双联体。我们的方法可以应用于其他类似的结构，并进一步优化，以处理更复杂的目标。
Cryo-electron microscopy (cryo-EM) has become the mainstream technique for studying macromolecular structures. Determining the structures of protein complexes is more accessible to structural biologists than ever before. Nevertheless, obtaining high-resolution structures of molecular motors like dynein is still an extremely challenging goal due to their troublesome behaviors in ice, their exceedingly flexible conformations, and their intricate architectures. Dynein is a large molecular machine that drives the movement of many essential cellular cargos and is also the key force generator that powers ciliary motility. High-resolution structural information of dyneins in different states is critical for the in-depth mechanistic understanding of their roles in cells. Here, we summarize the cryo-EM approaches that we have used to study the structures of outer-arm dynein arrays bound to microtubule doublets. Our approaches can be applied to other similar structures and further optimized to deal with even more complicated targets.