title: Structural analysis of muscle thin filament.
abstract: Thin sheets of Ac-Tm-Tn paracrystals were prepared in the presence of high concentration of Ca2+ ion and three-dimensional image analysis was performed. The optical diffraction pattern of an electron micrograph showed spots up to 1/1.6 nm-1 in the radial direction and up to 1/2.5 nm-1 in the axial direction, the best resolution ever obtained so far. The translationally filtered image showed clear polarity of filament which looked like a "spearhead" per each crossover repeat of actin helix. The three-dimensionally reconstructed model looked very similar to the inner regions (A+B domains) of the Ac-Tm-S1 complex obtained by Toyoshima and Wakabayashi (14, 15) when they were placed so that the "spearhead" pattern of the Tc-Tm-Tn complex and the "arrowhead" pattern of the Ac-Tm-S1 complex pointed in the same direction. The myosin-binding site of actin was identified by comparison of the two structures. The model of actin molecule cut out from the thin filament model had a low density region within itself, which was located about 2.5 nm from the helix axis. That low density region divided actin molecule into two domains, a large and a small domain. A dense "pillar" was detected which connected two neighboring actin molecules along a left-handed generic helix 1 nm from the helix axis. Two actin-actin binding sites which were responsible for the connection through the "pillar" were located on the inner surface of actin molecule. To obtain better crystalline arrays of actin, we tried a method utilizing adsorption to lipid. A positively-charged monolayer of lipids was formed on the surface of a small volume of buffer solution which was put in a microwell. Solution of negatively-charged F-actin was then injected into the buffer solution and was allowed to be joined to the lipid monolayer by electrostatic attraction. Fluidity of the lipid monolayer enabled the two-dimensional crystallization of actin. Electron microscopy revealed that larger paracrystalline arrays were formed more rapidly (less than 1 hr) than those formed within solution, which demonstrated the advantage of this adsorption method.
authors.affiliation: Faculty of Engineering, University of Tokyo, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Tomioka
authors.forename: H O
authors.initials: HO
authors.lastname: Ribi
authors.forename: M
authors.initials: M
authors.lastname: Tokunaga
authors.forename: T
authors.initials: T
authors.lastname: Furuno
authors.forename: H
authors.initials: H
authors.lastname: Sasabe
authors.forename: K
authors.initials: K
authors.lastname: Miyano
authors.forename: T
authors.initials: T
authors.lastname: Wakabayashi
mesh_terms: D000199:Actins / Q000737:chemistry / Q000648:ultrastructure*; D000818:Animals; D001665:Binding Sites; D002118:Calcium; D003461:Crystallography / Q000379:methods; D005583:Fourier Analysis; D007202:Indicators and Reagents; D008854:Microscopy, Electron / Q000379:methods; D008961:Models, Structural; D009132:Muscles / Q000648:ultrastructure*; D011487:Protein Conformation; D014335:Tropomyosin / Q000737:chemistry / Q000648:ultrastructure*
chemical_list: D000199:Actins; D007202:Indicators and Reagents; D014335:Tropomyosin; D002118:Calcium