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Y Shirakihara

Publications and source records attributed to Y Shirakihara.

8 recordsLinked to original sources
PubMed2000

Crystallization of the F41 fragment of flagellin and data collection from extremely thin crystals.

Flagellin, which constructs supercoiled filaments of the bacterial flagellum, is very difficult to crystallize because of its strong tendency to polymerize. We therefore crystallized the F41 fragment of flagellin, which does not polymerize because terminal regions that play important roles in polymerization are cleaved off. F41 was crystallized by the hanging drop vapor diffusion method in a mixture of polyethylene glycol, glycerol, and isopropanol, with a reservoir solution covered with silicon oil. The two key factors for success in growing sufficiently large crystals were isopropanol and silicon oil, which worked well to reduce the otherwise very high nucleation rate that resulted in hundreds of tiny crystals. The crystals were grown to very thin plates with thickness less than 10 microm, which made the collection of diffraction data very difficult. Freezing and annealing of the crystals and irradiation at synchrotron beamlines had to be carried out by specific methods and under specific conditions for its structure analysis at 2.0-A resolution.

Antigens, Bacterial↗
PubMed1999

[ATP synthase].

Explore the source record for details and available documents.

Catalytic Domain↗
PubMed1997

The crystal structure of the nucleotide-free alpha 3 beta 3 subcomplex of F1-ATPase from the thermophilic Bacillus PS3 is a symmetric trimer.

BACKGROUND: F1-ATPase, an oligomeric assembly with subunit stoichiometry alpha 3 beta 3 gamma delta epsilon, is the catalytic component of the ATP synthase complex, which plays a central role in energy transduction in bacteria, chloroplasts and mitochondria. The crystal structure of bovine mitochondrial F1-ATPase displays a marked asymmetry in the conformation and nucleotide content of the catalytic beta subunits. The alpha 3 beta 3 subcomplex of F1-ATPase has been assembled from subunits of the moderately thermophilic Bacillus PS3 made in Escherichia coli, and the subcomplex is active but does not show the catalytic cooperativity of intact F1-ATPase. The structure of this subcomplex should provide new information on the conformational variability of F1-ATPase and may provide insights into the unusual catalytic mechanism employed by this enzyme. RESULTS: The crystal structure of the nucleotide-free bacterial alpha 3 beta 3 subcomplex of F1-ATPase, determined at 3.2 A resolution, shows that the oligomer has exact threefold symmetry. The bacterial beta subunits adopt a conformation essentially identical to that of the nucleotide-free beta subunit in mitochondrial F1-ATPase; the alpha subunits have similar conformations in both structures. CONCLUSIONS: The structures of the bacterial F1-ATPase alpha and beta subunits are very similar to their counterparts in the mitochondrial enzyme, suggesting a common catalytic mechanism. The study presented here allows an analysis of the different conformations adopted by the alpha and beta subunits and may ultimately further our understanding of this mechanism.

Amino Acid Sequence↗
PubMed1994

Crystallization of 31 kDa C-terminal fragment of rat DNA polymerase beta.

A 248 residue C-terminal fragment of rat DNA polymerase beta (335 amino acid residues), a eukaryotic DNA repair enzyme, has been crystallized from polyethylene glycol 6000 solution. The crystals are orthorhombic, space group P2(1)2(1)2 with cell dimensions a = 120.3 A, b = 64.2 A, c = 39.4 A, and contain a single 31 kDa fragment in an asymmetric unit. The crystals diffract to 2.8 A resolution with laboratory X-ray source, and to 2.3 A resolution with synchrotron X-ray source, and are suitable for detailed structural analysis.

Animals↗
PubMed1991

Purification by dye-ligand chromatography and a crystallization study of the F1-ATPase and its major subunits, beta and alpha, from a thermophilic bacterium, PS3.

For a crystallization study, purification methods for F1-ATPase from a thermophilic bacterium, PS3, and its major subunits, beta and alpha, have been improved. The improvement depended on the introduction of dye-ligand chromatography columns to the previously adopted array of chromatography columns: a Blue-B (a blue dye bound to agarose) column was introduced for the F1 preparation, a Green-A column (a green dye attached to agarose) for the beta subunit, and a Blue-A (another blue dye, Cibacron Blue 3GA, bound to agarose) column for the alpha subunit. The improved preparations of all the proteins had purities of nearly 99%. Using the highly purified preparations of the proteins, crystallization conditions were searched for in a systematic way. Large plate crystals (0.2 X 0.5 X 0.5 mm) of F1 were grown from a polyethylene glycol solution. However, neither of the subunits was crystallized, in spite of extensive search for crystallization conditions.

Bacteria↗
PubMed1988

Crystal structure of the complex of phosphofructokinase from Escherichia coli with its reaction products.

The crystal structure of Escherichia coli phosphofructokinase complexed with its reaction products fructose 1,6-bisphosphate (Fru1,6P) and ADP/Mg2+, and the allosteric activator ADP/Mg2+, has been determined at 2.4 A resolution. The structure was solved by molecular replacement using the known structure of Bacillus stearothermophilus phosphofructokinase, and has been refined to a crystallographic R-factor of 0.165 for all data. The crystallization mixture contained the substrate fructose 6-phosphate, but the electron density maps showed clearly the presence of the product fructose 1,6-bisphosphate, presumably formed by the enzyme reaction with contaminating ATP. The crystal consists of tetrameric molecules with subunits in two different conformations despite their chemical identity. The magnesium ion in the "closed" subunit bridges the phosphate groups of the two products. In the "open" subunit, the products are about 1.5 A further apart, with the Mg2+ bound only to ADP. These two conformations probably represent two successive stages along the reaction pathway, in which the closure of the subunit is required to bring the substrates sufficiently close to react. This conformational change within the subunit is distinct from the quaternary structure change seen previously in the inactive T-state conformation. It is probably not involved in the co-operativity or allosteric control of the enzyme, since the co-operative product fructose 1,6-bisphosphate is not moved, nor are the subunit interfaces changed. The structure of the enzyme is similar to that of B. stearothermophilus phosphofructokinase, and confirms the location of the sites for the two reaction products (or substrates), and of the effector site binding the activator ADP/Mg2+. However, this structure gives a clearer picture of the active site, and of the interactions between the enzyme and its reaction products.

Adenosine Diphosphate↗
PubMed1984

Caulobacter crescentus flagellar filament has a right-handed helical form.

Caulobacter crescentus flagellar filaments were examined for their shape and handedness. Contour length, wavelength and height of the helical filaments were 1.34 +/- 0.14 micron, 1.08 +/- 0.05 micron and 0.27 +/- 0.04 micron, respectively. Together with the value of the filament diameter, 14 +/- 1.5 nm, the parameters of the curvature (alpha) and twist (phi) were calculated as 3.9(%) for alpha and 0.026 (rad) for phi, which are similar to those of the curly I filament of Salmonella typhimurium. Dark-field light microscopic analysis revealed that the C. crescentus wild-type filament possesses a right-handed helical form. Given the result that C. crescentus cells normally swim forward, in the opposite direction to a polar flagellum, it is likely that C. crescentus swims by rotation of a right-handed curly shaped flagellum in a clockwise sense, whereas S. typhimurium and Escherichia coli swim by rotation of left-handed normal type flagella in a counterclockwise sense.

Biopolymers↗