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purified recombinant enzyme free or with bound substrate L-Cys, hanging drop vapour diffusion method, mixing of 0.004 ml of 10 mg/ml protein solution with 0.004 ml of reservoir solution containing 18% w/v PEG 4000, 0.1 M potassium acetate, 0.05 M 2-(N-morpholino)ethanesulfonic acid, pH 6.0, 25°C, 2-7 days, X-ray diffraction structure determination and analysis at 2.38-2.82 A resolution
co-crystallization of L-cysteine and purified wild-type cysteine dioxygenase by the hanging drop, vapor-diffusion method at 16°C, the crystals grow as rods up to 0.2 x 0.2 x 0.8 mm in 1 week. Refinement of the crystal stucture leads to a final model with a crystallographic R-factor of 18.1% and a free R-factor of 21.5% at 2.7 A resolution.
enzyme with or without bound cysteine and formation of persulfenate, usage of crushed CDO crystals at 8 mg/mL in 10 mM Tris, pH 7.4, recrystallized by hanging drop vapour diffusion method, mixing of 0.0005 ml of crystal seed stock solution with 0.001 ml protein solution and 0.001 ml of reservoir solution containing 0.1 M trisodium citrate, pH 5.6, 24% PEG 4000, and 0.15 M ammonium acetate, final pH of 6.2, at room temperature, X-ray diffraction structure determination and analysis at pH 4.0-9.0 and 1.25-1.60 A resolution, overview
crystals are grown at 25°C by hanging-drop vapor-diffusion. The reservoir contains 20% methosylpolyethylene glycol 5000, 160 mM CaCl2, and 100 mM 2-morpholinjoethane-sulfonic acid (pH 6.5). Hanging drops consist of 2 microl of protein solution mixed with 2 microl of reservoir solution. Structure is solved to a nominal 1.75 A resolution.
electron paramagnetic study of substrate-O2 binding. Ordered binding of L-cysteine prior to NO and presumably O2. Upon addition of NO to cysteine dioxygenase in the presence of substrate L-cysteine, a low-spin(FeNO)7 signal with spin S of 1/2that accounts for 85% of the iron within the enzyme develops. Substitution of L-cysteine with isosteric substrate analogues cysteamine, 3-mercaptopropionic acid, and propane thiol does not produce any analogous signals.The unusual (FeNO)7, spin 1/2 electronic configuration adopted by the substrate-bound iron-nitrosyl cysteine dioxygenase is a result of the bidentate thiol/amine coordination of L-cysteine in the NO-bound active site
crystallization in sitting drops at 25°C using a reservoir of 0.1-0.25 M ammonium acetate, 0.1 M tri-sodium citrate, pH 5.6, with 22-26% (w/v) polyethylene glycol 4000. The co-crystals with 5 mM are grown using a reservoir of 0.15 M ammonium sulfate, 0.2 M sodium cacodylate, pH 6.5, with 26% (w/v) polyethylene glycol 8000. The crystal sturcture, solved by SAD phasing using selenomethionine-substituted protein, yields a final refined model with r = 18.0 and Rfree = 20.8 at 1.5-A resolution. Data from a co-crystallization experiment with 5 mM cysteine shows structural changes in the binding pocket, they are determined to 1.5 A resolution (final r = 19.8 and Rfree = 22.4).
generation of 21 CDO crystal structures at resolutions between 1.25 and 1.65 A. Of these, 16 are of C93A or Y157F CDO mutants either alone or bound to L-Cys, D-Cys, or the inhibitor homocysteine, the other five are of wild-type CDO bound to homocysteine, azide, or thiosulfate. Cys-soaked wild-type CDO crystals are analysed at pH 6.2 and pH 8.0, detailed overview
purified enzyme, hanging drop vapour diffusion method, mixing of 0.0015 ml of 7 mg/mL CDO mutant C93G in 10 mM sodium phosphate and 20 mM NaCl, pH 7.5, and 0.0006 ml of crushed wild-type CDO crystal seeds in their own growth solution consisting of 25% w/v PEG 1500, 13 mM succinate, 44 mM sodium phosphate, and 44 mM glycine, pH 5.2, with 0.0015 ml of reservoir buffer containing 26% w/v PEG 4000, 200 mM ammonium acetate, and 100 mM sodium citrate, pH 6.3, equilibration against reservoir solution, X-ray diffraction structure determination and analysis at 1.82 A resolution
purified recombinant enzyme complexed with cysteine persulfide or 3-mercaptopropionic acid persulfide, hanging drop vapor diffusion method, mixing of 0.0015 ml of 8 mg/ml protein with 0.0015 ml of reservoir solution containing 24-34% w/v PEG 4000, 100-250 mM ammonium acetate, 100 mM sodium citrate, pH 5.6, 0-4 mM dithionite, and 40 mM ligand, final pH is 6.1-6.2, 24°C, one week, X-ray diffraction structure determination and analysis at 1.63-2.05 A resolution
X-ray crystal structure