STRUCTURAL STUDIES OF THE MOTOR PROTEIN KINESIN BY IMAGE RECONSTRUCTION AND X-RAY CRYSTALLOGRAPHY. E. Mandelkow, F. Kozielski, A. Marx, E. Schonbrunn, S. Sack, V. Biou*, A. Thompson**, M. Thormahlen, E.-M. Mandelkow. Max-Planck-Unit for Struct. Mol. Biol., c/o DESY, D-22603 Hamburg, Germany; *Eur. Sync. Rad. Fac. (ESRF); **Eur. Mol. Biol. Lab. (EMBL), F-38042 Grenoble, France

Kinesin, a microtubule-activated ATPase, is a cytoplasmic motor which moves vesicles or organelles towards the distal end of microtubules. We have expressed several fragments of kinesin head domain in E. coli and used it for structural and biochemical studies of the microtubule-kinesin interaction. Open questions include: (a) Which tubulin subunit binds to kinesin? (b) What is the stoichiometry of binding? (c) How does the decoration by kinesin reveal the underlying microtubule lattice? (d) What is the polarity of the kinesin binding, and that of the tubulin subunits in a microtubule? Structural questions were addressed by image reconstruction of opened up microtubule walls decorated with several kinesin constructs, and by X-ray crystallography of the kinesin head domain. The reconstructions of the decorated and two-dimensionally ordered "tubulo- kinesin-complex" yields a low resolution (2nm) picture of how kinesin and tubulin interact with one another, including the tubulin lattice, binding stoichiometry, and polarity. Alternatively, the kinesin head can be induced to form well-ordered 3-dimensional crystals suitable for X-ray crystallography. The solution of the structure makes use of single isomorphous replacement including anomalous diffraction at different wavelengths, using a mercury derivative of kinesin. In addition, MAD data were obtained from derivatives in which methionines were replaced by seleno-methionines. The interpretation of the electron density will be presented.