title: Three-dimensional cryoelectron microscopy of 16-protofilament microtubules: structure, polarity, and interaction with motor proteins.
abstract: We present a three-dimensional (3D) map, reconstructed from electron microscope (EM) images of naturally occurring 16-protofilament (PF) microtubules (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 stained MTs decorated with kinesin in different nucleotide states. The comparison confirms that kinesin and ncd bind to identical sites and interact with both monomers 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 rearrangement. The relative polarity of undecorated and decorated 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 undecorated MTs. An image of tubulin PFs in zinc-induced sheets has been tentatively oriented by similar criteria.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: K
authors.initials: K
authors.lastname: Hirose
authors.forename: W B
authors.initials: WB
authors.lastname: Amos
authors.forename: A
authors.initials: A
authors.lastname: Lockhart
authors.forename: R A
authors.initials: RA
authors.lastname: Cross
authors.forename: L A
authors.initials: LA
authors.lastname: Amos
mesh_terms: D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D006135:Gryllidae; D016547:Kinesins / Q000737:chemistry; D008297:Male; D008854:Microscopy, Electron; D008870:Microtubules / Q000648:ultrastructure*; D008958:Models, Molecular; D013737:Testis / Q000737:chemistry; D014404:Tubulin / Q000737:chemistry
chemical_list: D014404:Tubulin; D000266:Adenylyl Imidodiphosphate; D016547:Kinesins