2.4.2.7 comparison of the crystal structure of PRPP-Mg2+-bound hAPRT to the ADE/PRPP-Mg2+ and AMP complex structures 759017 2.4.2.7 enzyme complexes phosphate-hAPRT, hypoxanthine-PRPP-Mg2+-hAPRT, IMP-hAPRT, and GMP-hAPRT, mixing of 400 nl of 5 mg/ml protein complexes in 20 mM Tris-HCl, pH 7.4, 5 mM MgCl2, with 200 nl of crystallization solution made of 85 mM Tris-HCl, pH 8.5, 170 mM NaOAc, 19-21% PEG 4000, and 0-30% glycerol, overnight at 20°C, X-ray diffraction structrue determination and analysis at resolution 1.55-1.90 A, molecular replacement using the structure with PDB ID 6FCH as template, and modeling 759493 2.4.2.7 enzyme in complex with adenosine-5'-monophosphate and a phosphate ion, crystallization at 4°C by hanging-drop vapor-diffusion method 658207 2.4.2.7 enzyme, 10 mg/ml, in complex with 9-deazaadenine and sulfate or Mg-phosphoribosyldiphosphate, 50 mM Hepes, pH 6.0, 8 mM MgCl2, 1 mM DTT, 1:2 molar ratio of 9-deazaadenine and iminoribitol, 1 mM sodium diphosphate, after 45 min incubation preparation of crystallization drops, crystals are obtained from mother liquid 0.1 M sodium acetate, pH 4.6, 24% polyethylene glycol 4000, 0.2 M ammonium sulfate, 0.05 M urea, 18°C, X-ray diffraction structure analysis, hydrogen bond network in the complexes 638168 2.4.2.7 four crystal structures: (1) a structure (the enzyme/Pi complex) refined at 2.4 A with inorganic phosphate or sulfate bound in the 5-phosphoribosyl binding pocket, (2) an adenine bound structure (the enzyme/adenine complex) refined at 2.4 A, which shows adenine together with phosphates both at the 5'-phosphoryl and PPi positions of the presumed PRPP binding site, (3) an AMP bound structure (the enzyme/AMP complex) refined at 2.4 A, and (4) an ADP bound structure (the enzyme/ADP complex), refined at 2.8 A containing the inhibitor ADP bound like AMP with both the alpha- and beta-phosphates occupying the 5'-phosphoribosyl binding site. No crystals of the enzyme in complex with 5-phosphoribosyl-alpha-1-pyrophosphate are obtained, likely because the enzyme catalyzes a slow breakdown of 5-phosphoribosyl-alpha-1-pyrophosphate to ribose 5-phosphate and PPi. The crystal structure suggests that the enzyme evolves from a 6-oxopurine phosphoribosyltransferase. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known adenine phosphoribosyltransferases implying that adenine phosphoribosyltransferase functionality in Crenarchaeotae has its evolutionary origin in this family of 6-oxopurine phosphoribosyltransferases. The N-terminal two-thirds of the polypeptide chain folds as a traditional type I PRTase with a five-stranded beta-sheet surrounded by helices. The C-terminal third adopts an unusual three-helix bundle structure that together with the nucleobase-binding loop undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site 729271 2.4.2.7 hanging drop vapour diffusion method, with 15% (v/v) glycerol, 25.5% (w/v) PEG 4000, 0.17 M sodium acetate, and 0.085 M Tris-HCl, pH 8.5 687844 2.4.2.7 mixing of protein solution 13-15 mg/ml with an equal volume of mother liquid 0.1 M Hepes, pH 7.5, 1.5 M lithium sulfate, then equilibration against mother liquid at 18°C, crystals appear after 3 days, X-ray diffraction structure analysis, also crystallization of the enzyme in presence of diphosphate, Mg2+ or inhibitor immucillin, which do not bind at the active site 638171 2.4.2.7 purified His-tagged recombinant enzyme in complex with inhibitors D-DIAB and L-DIAB, and also with adenine, X-ray diffraction structure determination and analysis of enzyme-inhibitor complexes at 1.78 A and 1.98 A resolution, respectively, modeling, structure comparisons 758562 2.4.2.7 purified recombinant enzyme APRT2, hanging-drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein in 200 mM NaCl and 20 mM Tris-HCl, pH 8.0, with 0.001 ml of precipitant solution containing 19% PEG 20000 and 0.1 M sodium citrate, pH 6.0, 30-50 days, 20°C, X-ray diffraction structure determination and analysis at 2.6 A resolution, molecular replacement using the structure of TthAPRT1 from Thermus thermophilus HB8 (PDB ID 1VCH) as a template 758925 2.4.2.7 purified recombinant enzyme in apoform and in complex with substrate adenine, sitting drop vapor diffusion method, mixing of 10 mg/ml protein in 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT, and 10% glycerol, with crystallization solutions (a) 0.1 M Tris/HCl pH 8.5, 30% PEG 4000 and 0.2 M MgCl2 and (b) 0.2 M sodium acetate trihydrate, 0.1 M Tris-HCl, pH 8.5, 30% PEG 4000, X-ray diffraction structure determination and analysis at 1.9-2.28 A resolution 759176