Preliminary crystallographic studies of two C-terminally truncated copper-containing nitrite reductases from Achromobacter cycloclastes: changed crystallizing behaviors caused by residue deletion

The C-terminal segment of copper-containing nitrite reductase from Achromobacter cycloclastes (AcNiR) has been found essential for maintaining both the quaternary structure and the enzyme activity of AcNiR. C-terminal despentapeptide AcNiR (NiRc-5) and desundecapeptide AcNiR (NiRc-11) are two important truncated mutants whose activities and stability have been affected by residue deletion. In this study, the two mutants were crystallized using the hanging drop vapor diffusion method. Crystals of NiRc-5 obtained at pH 5.0 and 6.2 both belonged to the P2(1)2(1)2(1) space group with unit cell parameters a=99.0 A, b=117.4 A, c=122.8 A (pH 5.0) and a=98.9A, b=117.7A, c=123.0A (pH 6.2). NiRc-11 was crystallized in two crystal forms: the tetragonal form belonged to the space group P4(1) with a=b=96.0A and c=146.6A; the monoclinic form belonged to the space group P2(1) with a=86.0A, b=110.1A, c=122.7A, and beta=101.9 degrees. The crystallizing behaviors of the two mutants differed from that of the native enzyme. Such change in combination with residue deletion is also discussed here.     

Preliminary X-ray crystallographic study of adenylosuccinate synthetase from Escherichia coli

Adenylosuccinate synthetase, a dimeric enzyme of 96,000 Mr, catalyzes the first committed step toward the de novo biosynthesis of AMP. Large, single crystals of adenylosuccinate synthetase from Escherichia coli grow from solutions of polyethylene glycol and ammonium sulfate. Crystals from ammonium sulfate belong to the orthorhombic space group P212121 with unit cell parameters a = 79.0 A, b = 70.2 A and c = 152.6 A. Crystals from polyethylene glycol belong to the space group P21, having unit cell parameters of a = 71.16 A, b = 71.99 A, c = 82.95 A, and beta = 71.52 degrees. The asymmetric units of both crystal forms probably contain the entire dimeric enzyme.     

Crystallization and preliminary X-ray analysis of the lactose-specific phosphocarrier protein IIAlac of the phosphoenolpyruvate: sugar phosphotransferase system from Staphylococcus aureus.

The IIA constituent of the lactose permease from Staphylococcus aureus has been crystallized in two different forms. Crystals of form I have been grown from polyethylene glycol 4000 with beta-octyl glucoside. They diffract to 3.0 A resolution and belong to space group C2 with unit cell dimensions a = 141.7 A, b = 130.7 A, c = 96.5 A and beta = 96.2 degrees. Form II crystals have been obtained from a solution containing polyethylene glycol 400, ammonium sulfate and manganese chloride. They diffract to at least 2.8 A resolution and belong to space group P2(1)2(1)2(1) with unit cell dimensions a = 89.9 A, b = 101.5 A and c = 90.9 A.     

Crystals of threonyl-tRNA synthetase from Thermus thermophilus. Preliminary crystallographic data

Crystals have been obtained of threonyl-tRNA synthetase from the extreme thermophile Thermus thermophilus using sodium formate as a precipitant. The crystals are very stable and diffract to at least 2.4 A. The crystals belong to space group P2(1)2(1)2(1) with cell parameters a = 61.4 A, b = 156.1 A, c = 177.3 A.     

Crystals of Anabaena PCC 7119 ferredoxin-NADP+ reductase

Crystals of ferredoxin-NADP+ reductase (FNR) from the cyanobacterium Anabaena PCC 7119 are grown in the presence of polyethylene glycol 6000 and beta-octyl glucoside. They belong to the hexagonal system. The cell parameters are a = b = 87.8 A, c = 92.7 A, space group P6(1) or P6(5), and a Vm of 3.0 A3/dalton for one molecule of 36,000 daltons per asymmetric unit. These crystals diffract strongly up to 1.9 A and are suitable for X-ray structural studies.     

Crystallization and preliminary X-ray study of blood coagulation factor IX/factor X-binding protein with anticoagulant activity from Habu snake venom.

Crystals of a blood anticoagulant from the venom of the Habu snake, Trimeresurus flavoviridis, have been obtained using ammonium sulfate by the vapor diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2 with cell dimensions a = 172 A, b = 86 A, c = 65 A, and diffract to at least 4.0 A resolution.     

Crystallization and initial X-ray analysis of the C2-subunit of crustacyanin.

Crystals of the C2-subunit of crustacyanin have been grown from solutions containing ammonium sulphate and 2-methyl-2,4-pentanediol as co-precipitants. The crystals belong to space group P2(1)2(1)2(1) (a = 42.0 A, b = 80.9 A, c = 110.8 A) with two subunits per asymmetric unit and diffract beyond 2.2 A resolution.     

Crystallization and preliminary X-ray study of pig lens aldose reductase

Crystals of pig lens aldose reductase have been grown from polyethylene glycol solutions at pH 6.2 and analysed by X-ray diffraction. Two crystal forms were obtained. The first belongs to space group P1 with unit cell dimensions a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees, gamma = 79.0 degrees, with four molecules in the unit cell related by a 222 non-crystallographic symmetry. The second crystal form is hexagonal. The space group is P6(2)22 with a = b = 101 A, c = 257 A and two molecules in the asymmetric unit. Both forms are suitable for X-ray structure analysis to better than 3 A resolution.     

Crystals of protein L1 from the 50 S ribosomal subunit of Thermus thermophilus. Preliminary crystallographic data

Crystals have been obtained of protein L1 from the large ribosomal subunit of an extreme thermophile. Thermus thermophilus, using a mixed solution of ammonium sulphate/methane pentanediol. The crystals belong to the space group P2(1)2(1)2, with cell parameters a = 82.7 A, b = 63.4 A, c = 44.7 A. They diffract X-rays to 2.3 A resolution.     

Crystallization and preliminary x-ray crystallographic study of NADH-cytochrome b5 reductase from pig liver microsomes

Crystals of the hydrophilic, catalytic domain (30 kDa) of pig liver NADH-cytochrome b5 reductase solubilized by the protease (cathepsin D) have been grown in a solution of polyethylene glycol by the vapor-diffusion procedure. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1) with unit-cell dimensions of a = 87.1, b = 73.2, and c = 49.0 A. The asymmetric unit contains one molecule of the enzyme. The x-ray diffraction patterns extend to 2.0-A resolution. On the other hand, the intact enzyme (35 kDa) containing the hydrophobic membrane-binding domain solubilized by the detergent (Triton N-101) has been crystallized also from the polyethylene glycol solution. The crystals are needle-shaped and still too small for x-ray diffraction study.     

Crystallization of the core protein of cellobiohydrolase II from Trichoderma reesei

Single crystals of the core protein of the cellulase cellobiohydrolase II have been grown in polyethylene glycol 6000 with the hanging drop method. Successful crystallization occurred only when 82 amino acids were removed from the N terminus by papain cleavage. Crystals belong to the space group P2(1) and have cell constants a = 49.1 A, b = 75.8 A, c = 92.9 A, beta = 103.2. The diffraction pattern extends to better than 2.0 A.     

Crystallization and preliminary crystallographic studies of the FAD domain of corn NADH: nitrate reductase.

Crystals of the flavin domain of corn nitrate reductase expressed in Escherichia coli have been obtained at room temperature, using sodium citrate as precipitant. The crystals diffract to at least 2.5 A resolution at a synchrotron radiation source. Precession photographs show that they belong to the rhombohedral space group R3 with unit cell dimensions a = b = 145.4 A, c = 47.5 A, alpha = beta = 90 degrees and gamma = 120 degrees. There is one subunit per asymmetric unit which gives a packing density of 3.2 A3/Da, indicating a high solvent content in these crystals.     

Crystallization and preliminary crystallographic study of stonustoxin, a protein lethal factor isolated from the stonefish (Synanceja horrida) venom

Crystals of stonustoxin have been obtained and diffract to 3.4 A resolution. Stonustoxin is a protein lethal factor isolated from the venom of the stonefish, Synanceja horrida. The crystals belong to the tetragonal space group P422, with unit cell constants a = b = 109.0 A, c = 245.7 A. A native stonustoxin molecule has two subunits, designated alpha and beta, respectively, and there is one stonustoxin molecule per asymmetric unit.     

Plasmodium falciparum ARFGAP: expression and crystallization of the catalytic domain

GTPase activating protein for ARF GTPAse (ARFGAP) from the malaria parasite Plasmodium falciparum was expressed, purified and crystallized. Crystals of ARFGAP belong to trigonal space group P321 (or its enantiomorph) with unit cell parameters a=b=95.89 and c=92.46 A. Diffraction data to 2.4-A resolution have been collected. Calculation of self-rotation function suggested the presence of two molecules in the asymmetric unit.     

Crystals of the NC1 domain of human type IV collagen

Crystals of the non-collagenous C-terminal region (NC1) of type IV collagen have been obtained from human placenta. These crystals diffract to 2.0 A, and belong to space group P22(1)2(1), with cell dimensions a = 81 A, b = 158 A, c = 138 A, alpha = beta = gamma = 90 degrees. The crystals contain one hexamer in the asymmetric unit; they are very stable with respect to X-rays.     

Crystallization and X-ray crystallographic studies of an inhibitor from rye (Secale cereale) active against Acanthoscelides obtectus and Zabrotes subfasciatus alpha-amylases

Crystals of a new inhibitor present in rye seeds active against alpha-amylases from crop pests Acanthoscelides obtectus and Zabrotes subfasciatus have been obtained. A native dataset was collected at 2.21 A resolution with 99.3% completeness at CPr beamline at LNLS. The crystals belong to the trigonal system, space group P3(1)21 with a=b=78.21 A, and c=59.61 A. The crystal calculated solvent content is compatible with one dimer per asymmetric unit.     

X-ray grade crystals of the enzymatic fragment of diphtheria toxin

The enzymatic fragment of diphtheria toxin, fragment A (Mr = 21,167), complexed to the dinucleotide adenosine 3',5'-uridine (ApU), has been crystallized at two different values of pH by hanging drop vapor diffusion. Crystals grown at a pH value of 5.0 (from I) belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 71.2 A, b = 73.0 A, c = 139.8 A and four protomers in the asymmetric unit. Crystals grown at a pH value of 8.1 (form II) belong to the monoclinic space group C2, with unit cell parameters a = 65.2 A, b = 85.6 A, c = 34.6 A, beta = 103.0 degrees and one protomer in the asymmetric unit. Both crystal forms diffract to 2.5 A resolution. The molecular structures of fragment A obtained from these two crystal forms may illuminate the pH-dependent transition of diphtheria toxin during membrane translocation.     

Preliminary crystallographic analysis of cardiotoxin V with major fusion activity from Taiwan cobra (Naja naja atra) venom

Crystals of a cardiotoxin from Taiwan cobra venom have been obtained by the vapor diffusion method using methyl pentanediol as precipitant. The crystals belong to the hexagonal space group P6(1)22 (or P6(5)22), with cell dimensions a = b = 47.5 A, c = 111.3 A, alpha = beta = 90 degrees and gamma = 120 degrees and diffract to a resolution of 2.2 A. There is one molecule per asymmetric unit and the solvent content is estimated to be 53%.     

Crystallization and crystal data on tyrosine phenol-lyase

Crystals of the apoenzyme of tyrosine phenol-lyase (EC 4.1.99.2), a pyridoxal 5'-phosphate-dependent enzyme from Citrobacter intermedius, have been grown by vapor diffusion of an ammonium sulfate solution to a protein solution. The crystals belong to space group P2(1)2(1)2, with dimensions of a = 75.5 A, b = 138.4 A and c = 94.1 A and diffract up to 2.7 A resolution. The asymmetric unit contains one half of the enzyme tetrameric molecule. Two heavy-atom derivatives of the crystals have been obtained.     

Preliminary X-ray studies on the GTP: AMP phosphotransferase from beef heart mitochondria

Crystals of GTP: AMP phosphotransferase from beef heart mitochondria suitable for X-ray analysis have been grown. They belong to space group I4 with unit cell dimensions: a = b = 88.2 A, c = 147.8 A. The asymmetric unit contains two molecules each of Mr = 26,000. So far, two heavy-atom derivatives have been obtained.