title: Two-dimensional crystallization and analysis of projection images of intact Thermus thermophilus V-ATPase.
abstract: H(+)-ATPase/synthases are membrane-bound rotary nanomotors that are essential for energy conversion in nearly all life forms. A member of the family of the vacuolar-type ATPases (V-ATPases) from Thermus thermophilus, sometimes also termed A-type ATPase, was purified to homogeneity and subjected to two-dimensional (2D) crystallization trials. A novel approach to the 2D crystallization of unstable complexes yielded densely packed sheets of V-ATPase, exhibiting crystalline arrays. Aggregation of the V-ATPase under acidic conditions during reconstitution circumvented the continuous dissociation of the whole complex into the V(1) and V(o) domains. The resulting three-dimensional aggregates were converted into 2D sheets by the use of a basic buffer, and after a short annealing cycle, ordered arrays of up to 1.5 microm diameter appeared. Fourier transforms calculated from micrographs taken from the negatively stained sample showed diffraction spots to a resolution of 23A. The Fourier transforms of the untilted images revealed unit-cell dimensions of a=232A, b=132A, and gamma=90 degrees , and a projection map was calculated by merging 11 images. The most probable molecular packing suggests p22(1)2(1) symmetry of the crystals and dimer contacts between the V(1) domains.
authors.affiliation: Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Christoph
authors.initials: C
authors.lastname: Gerle
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Tani
authors.forename: Ken
authors.initials: K
authors.lastname: Yokoyama
authors.forename: Masatada
authors.initials: M
authors.lastname: Tamakoshi
authors.forename: Masasuke
authors.initials: M
authors.lastname: Yoshida
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Fujiyoshi
authors.forename: Kaoru
authors.initials: K
authors.lastname: Mitsuoka
mesh_terms: D003460:Crystallization; D005583:Fourier Analysis; D006863:Hydrogen-Ion Concentration; D008855:Microscopy, Electron, Scanning; D008956:Models, Chemical; D016963:Thermus thermophilus / Q000201:enzymology*; D025262:Vacuolar Proton-Translocating ATPases / Q000032:analysis* / Q000737:chemistry / Q000648:ultrastructure
chemical_list: D025262:Vacuolar Proton-Translocating ATPases