The project direction in 1996 has been the completion of the three dimensional structures of the motor domains of the NCD and kinesin. In early 1996, crystals were obtained for a mutant kinesin, Gly234Ala, which binds tightly to microtubules, but does not hydrolyze ATP. The mutation is of a universally conserved glycine that, in the homologs myosin and G proteins, participates in binding and sensing of the g phosphate of ATP. X-ray data on the mutant were collected at CHESS beam line F1 (l=0.908 ?). The mutant with MgADP bound shows no significant structural changes (C. Sindelar, J. Kull, et al., manuscript in preparation). The coordinates are being refined. We also attempted to crystallize the motor domains of kinesin and NCD in alternate, nucleotide-induced, conformational states (with ATPg-S, ADPNP or ADPCPbound). No diffraction quality crystals are obtained for kinesin with these nucleotides so far. The NCD motor domain has been crystallized in the presence of AMPPCP, but the crystals are still too small for X-ray diffraction (20 x 10 x 10 mm) Most effort has been directed to obtaining crystals of dimeric forms of kinesin and NCD in different conformational states. Crystals are obtained for three different constructs of NCD, though all of them show weak diffraction. The best data (to 4.5? with Rmerge 8.6%) were collected on a flash-frozen crystal of the NCD dimer D250-K700 using RAXIS II image plate detector. The crystals belong to the monoclinic space group P21 with unit cell dimensions a=153.4?, b=201.4 ?, c=195.3? and b=90.5o. Attempts to obtain protein phases for the NCD dimer with iodo-modified nucleotides failed because of weak diffraction from the crystals and iodines. Molecular replacement solutions to fit the dimer structure are ambiguous, though encouraging. The NCD dimer was also crystallized in an alternate nucleotide state induced by AMPPCP. These crystals are still too small (10 x 10 x 5 mm) for X-ray diffraction. We are searching for new crystals forms obtained under different conditions or with alternate constructs.
Showing the most recent 10 out of 375 publications
