Structural investigations into the atomic details of the kinesin walking mechanism and their interaction with microtubules by combining dada from 3D Cryo-electron microscopy and X-ray crystallography

通过结合 3D 冷冻电子显微镜和 X 射线晶体学的数据，对驱动蛋白行走机制的原子细节及其与微管的相互作用进行结构研究

• 批准号: 5240990
• 负责人: Professor Dr. Andreas Hoenger
• 金额: --
• 依托单位: Department of Molecular, Cellular and Developmental Biology (MCDB)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2000
• 资助国家: 德国
• 起止时间: 1999-12-31 至 2004-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5240990?language=en
• 关键词: Structural; investigations; into; atomic; details

Using cryo-electron microscopy and digital image analysis, the structures of several kinesin-microtubule complexes have been solved recently in our lab and others to a resolution of approximately 20Å. Within the same time the atomic resolution structures of kinesin motor head domains (using X-ray crystallography) and the tubulin ab-heterodimer (using electron crystallography) have been solved as well. Together with a large amount of biochemical and biophysical data these components can now be assembled to gain a comprehensive insight in the structural and functional properties of kinesin-like molecular motors. To this end, however, it will be necessary to further develop methods, which allow to accurately merge structural data from different sources such as X-ray or EM. In addition, we will have to refine and modify the currently available EM-based image reconstruction methods for some specific applications. Concerning EM-reconstructions of microtubule-motor complexes we are now at a point where the conventional reconstruction methods cannot be readily applied anymore. Helical 3-D averaging cannot be used to accurately reconstruct the binding and walking properties of dimeric kinesin molecules since the binding pattern of kinesin dimers during stepping involves two binding sites on the microtubule surface. It is very unlikely that during random decoration of the microtubule surface these patterns will ever follow any helical symmetry. This proposal aims 1: at the development of methods which will allow us to accurately reconstruct the binding conformation of dimeric kinesin head domains on the microtubule surface, and 2: to improve the interpretation of the molecular processes combining biochemical data with structural information of the entire complexes at intermediate, and their individual components at high resolution.

利用低温电子显微镜和数字图像分析技术，最近在我们的实验室和其他实验室解决了几个动蛋白-微管复合体的结构，分辨率约为20？同时，还解决了运动头区(用X射线结晶学)和微管蛋白异质二聚体(用电子结晶学)的原子分辨结构。结合大量的生化和生物物理数据，这些组件现在可以组装在一起，以全面了解激动素类分子马达的结构和功能特性。然而，为此目的，有必要进一步开发方法，使之能够准确地合并来自不同来源的结构数据，例如X射线或EM。此外，我们还需要对现有的基于EM的图像重建方法进行改进和修改，以适应一些具体的应用。关于微管-运动复合体的EM重建，我们现在处于一个点上，传统的重建方法不再容易地应用。由于运动蛋白二聚体在步进过程中的结合模式涉及微管表面的两个结合部位，因此螺旋三维平均不能用于准确重建二聚体运动蛋白分子的结合和行走特性。在微管表面的随机装饰过程中，这些图案不太可能遵循任何螺旋对称。这项建议的目的1：发展方法，使我们能够准确地重建微管表面二聚体运动蛋白头部结构域的结合构象；2，提高对分子过程的解释，将生化数据与整个中间体及其单个组分的结构信息结合在一起，以高分辨率进行解释。