STRUCTURAL STUDIES ON MICROTUBULE MOTORS

这项工作的长期目标是了解其作用机制 与微管相互作用的机械力化学酶。目标 这方面的应用主要是使用冷冻电子显微镜和图像分析 建立中分辨率微管三维信息数据库 以运动素和NCD的运动域装饰存在和 不含非水解性三磷酸腺苷类似物。这两个运动域有 类似的序列，但在微管上以相反的方向传播。这个 获得的3D图将显示这些马达如何与微管相互作用 并应提供对构象变化的洞察力 发生在这些功能不同的分子的运动域中 在他们的依恋周期中。此外，黄金集群标签将是 用于定位发动机上的表面残留物。 获得的数据将补充正在收集的高分辨率数据 在其他实验室的微管蛋白锌片上(伯克利唐宁)和3- 运动域的D晶体(Vale&Fletterick，UCSF；Goldstein， 加州大学圣迭戈分校)。电机-轨道复合体和高分辨率电磁数据的中等分辨率 来自其他组件的单个组件的分辨率x射线和EM图 这两个实验室都将是建立发动机原子模型的关键 附着在微管上。这种方法的组合已经非常 成功研究肌动球蛋白系统。 这项工作属于基础生物医学研究的范畴，因为它 不是针对特定的疾病，而是具有基本的和根本的 与健康和患病的州都有关联。在这里获得的数据 将提供对细胞内和轴突机制的深入了解 运输，以及细胞分裂过程中的染色体运动。归根结底， 数据可能被证明对理解这些正常的疾病很重要 细胞过程是异常的。

The long-term goals of the work are to understand the mechanism of action of the mechanochemical enzymes which interact with microtubules. The goals of this application are to use cryo-electron microscopy and image analysis to build a database of moderate resolution 3-D information on microtubules decorated with the motor domains of kinesin and NCD in the presence and absence of a non-hydrolyzable ATP analogue. These two motor domains have similar sequences but travel in opposite directions on microtubules. The 3-D maps obtained will show how these motors interact with the microtubule protofilaments and should provide insights into the conformational changes occurring in the motor domain of these functionally distinct molecules during their attachment cycle. In addition, gold cluster labelling will be used to localize surface residues on the motors. The data obtained will complement the high resolution data being collected in other laboratories on zinc sheets of tubulin (Downing, Berkeley) and 3- D crystals of the motor domains (Vale & Fletterick, UCSF; Goldstein, UCSD). Moderate resolution EM data on the motor-track complex and the high resolution x-ray and EM maps of the individual components from other laboratories will both be essential for building an atomic model of motors attached to microtubules. Such a combination of approaches has been very successful in studying the actomyosin system. This work falls into the category of Basic Biomedical Research in that it is not targeted to a particular disease but has a basic and fundamental relevance for both the healthy and diseased states. The data obtained here will provide insights into the mechanisms of intracellular and axonal transport, and chromosome movements during cell division. Ultimately, the data may prove important for understanding diseases where these normal cellular processes are aberrant.