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

利用低温电子显微镜和数字图像分析，几个驱动蛋白微管复合物的结构已经解决了最近在我们的实验室和其他人的分辨率约为20 μ m。在同一时间内，驱动蛋白马达头域（使用X射线晶体学）和微管蛋白ab-异源二聚体（使用电子晶体学）的原子分辨率结构也得到了解决。与大量的生物化学和生物物理数据一起，这些组件现在可以组装，以获得驱动蛋白样分子马达的结构和功能特性的全面了解。然而，为此，有必要进一步开发方法，以便准确地合并来自不同来源（如X射线或EM）的结构数据。此外，我们将不得不完善和修改目前可用的EM为基础的图像重建方法，一些特定的应用。关于微管-运动复合体的EM重建，我们现在处于传统重建方法不能再容易应用的点。螺旋3-D平均不能用于准确地重建二聚体驱动蛋白分子的结合和行走特性，因为在步进过程中驱动蛋白二聚体的结合模式涉及微管表面上的两个结合位点。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性。该提案的目标是：在开发的方法，这将使我们能够准确地重建微管表面上的二聚体驱动蛋白头部结构域的结合构象，和2：以改善结合生化数据与结构信息的整个复合物在中间的分子过程的解释，和他们的个别组件在高分辨率。

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.