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

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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.