Three-dimensional cryo-electron microscopy of dimeric kinesin and ncd motor domains on microtubules

We present a three-dimensional (3D) map, recon-structed from electron microscope (EM) images of naturally occurring 16-protofilament (PF) microtu-bules (MTs) in ice. We compare it with the tubulin in six 3D maps of MTs decorated with motor domains, three from frozen MTs decorated with kinesin or ncd in the tightly bound AMP–PNP state, and three from negatively stainedMTs decorated with kinesin in different nucleotide states. The comparison con-firms that kinesin and ncd bind to identical sites and interactwith bothmonomers of a tubulin dimer. Maps of specimens in negative stain and in ice are similar except that the protein in the top half of a motor domain appears denser in negative stain. The interactions have only a small effect on tubulin structure; the outward appearance is unchanged, but there seems to be a small internal rearrange-ment. The relative polarity of undecorated anddeco-rated MTs is evident from their 3D structures. This agrees with the absolute polarities indicated by the orientations of motors in decorated specimens and by polar superposition patterns calculated for un-decorated MTs. An image of tubulin PFs in zinc-induced sheets has been tentatively oriented by similar criteria. r 1997 Academic Pres