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.
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