Crystallization and preliminary X-ray investigation of bovine liver mitochondrial aldehyde dehydrogenase.

Aldehyde dehydrogenase from bovine liver mitochondria has been crystallized using the sitting drop method of vapor diffusion at 22 degrees C. The crystals formed from solutions containing, 40 mM-sodium citrate, 1 mM-NAD+ and 21% to 24% polyethylene glycol 3400 (pH 5.3 to 5.5). X-ray diffraction data collected from these crystals indicate that the crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 153.7 A, b = 159.37 A and c = 101.45 A. The crystals diffract to at least 2.9 A and a tetramer may comprise the asymmetric unit.     

Crystallization of and preliminary crystallographic data for the N-terminal half-molecule of ovotransferrin

The N-terminal half molecule of ovotransferrin has been crystallized from a polyethylene glycol 6000 solution by means of the vapor diffusion method. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with cell dimension of a = 47.0, b = 90.2, and c = 76.2 A. The crystals diffract X-rays to a resolution limit of at least 2.0 A and are resistant to X-ray radiation damage. They appear to be suitable for X-ray structure analysis.     

Crystallization and preliminary X-ray study of horse pancreatic lipase

Horse (Equus caballus) pancreatic lipase (EC 3.1.1.3) has been crystallized using the hanging drop method of vapour diffusion at 20 degrees C. The best crystals were grown from an 8 mg/ml solution in 10 to 20% (w/v) polyethylene glycol 8000, 10 mM-MgCl2, 0.1 M-NaCl, 0.1 M-Mes buffer (pH 5.6). They reach dimensions of 0.8 mm x 0.4 mm x 0.6 mm. X-ray examination of the lipase crystals shows that they are orthorombic with a space group P2(1)2(1)2(1). Their cell dimensions are a = 79.8 A, b = 97.2 A c = 145.3 A. Two molecules per asymmetric unit give a Vm value of 2.82 A3/dalton (56% water content). Lipase crystals strongly diffract to at least 1.8 A resolution. Some molecular properties of horse lipase compared to those of the better-known porcine enzyme are also presented.     

Crystallization and preliminary X-ray crystallographic studies of Thermus thermophilus HB8 MutM protein involved in repairs of oxidative DNA damage

MutM protein, which removes the oxidatively damaged DNA base product, 8-oxoguanine (GO), has been crystallized by means of a hanging-drop vapor-diffusion procedure using polyethyleneglycol monomethylether 2000 as a precipitant in 2-(cyclohexylamino) ethanesulfonic acid (CHES) buffer, pH 9.8. The diffraction data derived from oscillation photographs indicate that the crystals belong to the monoclinic system and space group P2(1). The crystals have unit-cell dimensions of a = 45.4 A, b = 62.0 A, c = 99.7 A, and beta = 90.8 degrees. Assuming that the asymmetric unit contains two molecules, the Vm value was calculated to be 2.35 A(3).Da(-1). The crystals diffracted X-rays to at least 2.1 A resolution and were suitable for high-resolution X-ray crystal structure determination.     

Preliminary crystallographic study of glucose-6-phosphate dehydrogenase from rat liver

Crystals of D-glucose-6-phosphate: NADP+ oxidoreductase were obtained with the hanging drop, vapor diffusion and batch methods from ammonium sulfate-containing solutions. X-ray diffraction photographs indicate that the crystals belong to the orthorhombic space groups I222 or I2(1)2(1)2(1) with unit cell dimensions of a = 66.0 A, b = 140.8 A and c = 177.8 A. These data, together with results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crystal density experiments, indicate that there is one 116,000 Mr dimer per asymmetric unit. The crystals diffract to at least 2.2 A and are suitable for X-ray crystallographic structure determination.     

A crystal form of ribulose-1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum in the activated state

A new crystal form of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from Nicotiana tabacum has been obtained at alkaline pH with polyethylene glycol 8000 in the presence of a non-ionic detergent, beta-octyl glucoside. The crystals are grown at room temperature by the hanging-drop vapor diffusion technique from a protein solution containing enzyme complexed with CO2, Mg2+, and the transition state analog 2-C-carboxy-D-arabinitol-1,5-bisphosphate. The crystals belong to the the space group P3(1)21 (or P3(2)21) with the cell parameters a = 204.6 A, and c = 117.4 A (1 A = 0.1 nm). The asymmetric unit contains half (L4S4: L, large subunit, 53,000 Mr; S, small subunit, 15,000 Mr) of a hexadecameric molecule (L8S8, 540,000 Mr). The crystals diffract to at least 2.6 A Bragg spacing and are suitable for X-ray structure determination.     

Crystallization of the calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli K12

Single crystals of phosphoenolpyruvate carboxykinase from Escherichia coli K12 have been grown in the orthorhombic crystal system. Single crystals developed to a maximum size of 0.25 mm x 0.25 mm x 1.5 mm by the technique of washing and reseeding. The space group is P2(1)2(1)2(1), with a = 77.24 A, b = 89.18 A, c = 93.24 A and Z = 4; there is one enzyme molecule per crystallographic asymmetric unit and the solvent content is estimated to be 59%. The crystals diffract to at least 2.8 A d spacings and decompose in the X-ray beam after approximately two days of exposure.     

Crystallization of and preliminary crystallographic data for Bacillus stearothermophilus cyclodextrin glucanotransferase

Cyclodextrin glucanotransferase from Bacillus stearothermophilus TC-91 has been crystallized from an ammonium sulfate solution by the dialysis equilibrium method. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with cell dimensions of a = 125.5 A, b = 88.1 A, and c = 81.5 A. The crystals appear to be suitable for X-ray structure analysis, diffracting to at least 2.1 A and being resistant to radiation damage.     

Preliminary X-ray diffraction study of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

Crystals from the dimeric enzyme ribulose-1,5-bisphosphate carboxylase of the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. The crystals are of the quarternary complex comprising enzyme: activator CO2 (as a carbamate): Mg2+: 2- carboxyarabinitol -1,5-bisphosphate (as a transition state analog). X-ray diffraction photographs show symmetry consistent with space group P4(1)2(1)2 or the corresponding enantiomorphic space group. Cell parameters are a = b = 82 A, c = 324 A with two subunits per asymmetric unit. The crystals diffract to at least 3 A resolution.     

Crystallization of the Bacillus subtilis histidine-containing phosphocarrier protein HPr and of some of its site-directed mutants

The histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been crystallized. Two of the site-directed mutants aimed at probing function produce crystals suitable for X-ray studies. The mutant in which His15 is substituted by an alanyl residue crystallizes from ammonium sulfate solution in space group P3(1)21 or P3(2)21, with unit cell dimensions: a = b = 47.3 A; c = 61.5 A. These crystals diffract to at least 1.8 A resolution. The mutant in which Ser46 is substituted by an aspartyl residue crystallizes from polyethylene glycol 4000 solution in space group P2(1), with unit cell dimensions: a = 49.4 A; b = 25.6 A; c = 60.3 A; beta = 109 degrees. These crystals diffract to at least 2.0 A resolution.     

Crystallization of the DpnM methylase from the DpnII restriction system of Streptococcus pneumoniae

Three proteins, two DNA methylases and an endonuclease, from the DpnII restriction system of Streptococcus pneumoniae recognize the DNA sequence 5' GATC 3' but have very different amino acid sequences, which make them interesting subjects for structural determination. A purification procedure was developed that conveniently yields milligram amounts of the DpnM methylase. The DpnM protein tends to precipitate at reduced ionic strength, and this property was exploited to yield well-formed bipyramidal crystals. By X-ray diffraction, the crystals of DpnM were found to be orthorhombic, with cell dimensions a = 56.9 A, b = 68.2 A, c = 84.5 A; systematic absences identify the space group as P2(1)2(1)2(1). Diffraction extends beyond 3 A, so the crystals may allow structural determination at atomic resolution.     

Crystallization, preliminary X-ray study and crystal activity of the hydrogenase from Desulfovibrio gigas

Hydrogenase (EC 1.12) from Desulfovibrio gigas is a dimeric enzyme (26 and 62 (X 10(3) Mr) that catalyzes the reversible oxidation of molecular hydrogen. Single crystals of hydrogenase have been produced using the hanging drop method, with either PEG (polyethylene glycol) 6000 or ammonium sulfate as precipitants at pH 6.5. X-ray examination of the crystals indicates that those obtained with ammonium sulfate are suitable for structure determination to at least 3.0 A resolution when synchrotron radiation Sources are used (1 A = 0.1 nm). The crystals are monoclinic, with space group C2, and cell dimensions a = 257.0 A, b = 184.7 A, c = 148.3 A and beta = 101.3 degrees, and contain between four and ten molecules per asymmetric unit. The enzyme can be reactivated within the crystals under reducing conditions without crystal damage.     

Crystallization and preliminary X-ray studies of D-serine dehydratase from Escherichia coli

Single crystals of D-serine dehydratase from Escherichia coli complexed with 3-amino-2-hydroxypropionate have been obtained from ammonium sulfate solution (pH 7.0) by vapor diffusion. The crystals belong to the trigonal space group P3(1) or P3(2) with a = b = 81.3 A and c = 58.4 A. The asymmetric unit cell contains one protein molecule with Mr = 48,289. The crystals diffract to at least 3.0 A resolution and are suitable for X-ray structure analysis.     

Studies on the hydrolyzing mechanism for cyclodextrins of Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII). X-ray structure of the mutant E354A complexed with beta-cyclodextrin, and kinetic analyses on cyclodextrins

Crystals of the mutant E354A of Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII) complexed with beta-cyclodextrin were prepared by a soaking method, and the diffraction data were collected at 100 K, using Synchrotron radiation (SPring-8). The crystals belong to an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions a = 111.1 A, b = 117.7 A, c = 113.3 A, which is almost isomorphous with crystals of the wild-type TVAII, and the structure was refined to an R-factor = 0.208 (R(free) = 0.252) using 3.0 A resolution data. The refined structure shows that the interactions between Phe286 and two C6 atoms of beta-cyclodextrin at the hydrolyzing site are important for TVAII to recognize cyclodextrins as substrates. This observation from the X-ray structure was supported by kinetic analyses of cyclodextrins using the wild-type TVAII, the mutant F286A and F286L. These studies also suggested that the TVAII-hydrolyzing mechanism for cyclodextrins is slightly different from that for starch.     

Crystallization and preliminary X-ray diffraction studies of a peroxidase from barley grain.

Crystals suitable for X-ray diffraction analysis of both glycosylated and non-glycosylated forms of a barley peroxidase have been grown. The crystals of the glycosylated peroxidase have been grown by the hanging drop vapour diffusion method using polyethylene glycol 4000 as the precipitant in the presence of n-propanol and potassium iodide at pH 8.5. The crystals are needles belonging to the orthorhombic spacegroup P2(1)2(1)2(1) with unit cell dimensions a = 62.95 A, b = 66.24 A and c = 70.78 A. There is one barley peroxidase molecule in the asymmetric unit. The crystals contain approximately 38% solvent and appear to be stable to lengthy X-ray exposure. They diffract to beyond 1.9 A.     

Crystallization and preliminary X-ray studies of two isolectins from the seeds of Lathyrus ochrus

Two isolectins from the seeds of Lathyrus ochrus, LOL I and LOL II, which specifically bind N-acetyllactosamine, have been crystallized using the hanging-drop method and the interface diffusion method, respectively. In the case of LOL I, 2-methylpentane-2,4-diol, polyethylene-glycol 400 or ammonium sulphate have been used as precipitating agents. The best crystals of LOL I were grown at room temperature from a solution of 40% (v/v) methylpentane diol, 50 mM-Hepes at pH 7.5. LOL II crystals have been grown at room temperature from a solution of 32% (v/v) methylpentane diol, 50 mM-2-(N-morpholino)-ethanesulphonic acid at pH 5.5. X-ray examination of the LOL I and LOL II crystals shows that both are monoclinic, space group P2(1). Their cell dimensions are: LOL I, a = 56.4 A, b = 138.8 A, c = 62.9 A, beta = 91 degrees; and LOL II, a = 54.8 A, b = 71.4 A, c = 105.5 A, beta = 105 degrees. Density measurements of the crystals of LOL I indicate that there are two molecules per asymetric unit (Vm = 2.07 A3/dalton). LOL I crystals diffract strongly up to at least 1.8 resolution. Putative crystals of complexes of LOL I with various glycosides were obtained through co-crystallization under the conditions used for the native protein.     

Preliminary crystallographic study of a pseudoazurin from methylotrophic bacterium, Methylobacterium extorquens AM1

Single crystals of pseudoazurin, one of the blue copper proteins produced by methylotrophic bacterium Methylobacterium extorquens AM1, have been obtained by the method of vapor diffusion with ammonium sulfate as a precipitant at pH 8.0. Crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 52.619(4) A, b = 63.280(6) A, c = 35.133(4) A. The asymmetric unit includes one molecule of pseudoazurin (Vm = 2.18 A3/dalton). The crystals are so stable against X-ray irradiation that diffraction intensities of the native crystal up to 1.68 A resolution could be collected from only one crystal. Among the many heavy-metal reagents examined, uranyl acetate gave an effective isomorphous derivative.     

Crystallization and a preliminary X-ray crystallographic study of alpha-amylase from Bacillus licheniformis

alpha-Amylase from Bacillus licheniformis has been crystallized by the hanging-drop vapor diffusion method in the presence of calcium ions using ammonium sulfate as precipitant. The crystals are tetragonal, belonging to the space group P4(1)2(1)2 (or P4(3)2(1)2), with unit cell dimensions of a = 119.9 and c = 85.4 A. The asymmetric unit contains one molecule of alpha-amylase, with a crystal volume per protein mass (VM) of 2.78 A3/Da. The crystals diffract to better than 2.0 A Bragg spacing when exposed to synchrotron X-rays and they are reasonably stable in the X-ray beam. Thus the crystals are suitable for structure determination at high resolution by X-ray methods.     

Crystallization of the soluble lytic transglycosylase from Escherichia coli K12

Lytic transglycosylases degrade the murein polymer of the bacterial cell wall to 1,6-anhydromuropeptides. These enzymes are of significant medical interest, not only because they are ideal targets for the development of new classes of antibiotics, but also because the low molecular weight products of their catalytic action can cause diverse biological activities in humans, which can be either beneficial or toxic. A soluble lytic transglycosylase was purified from an overproducing Escherichia coli strain and X-ray quality crystals were obtained at room temperature from hanging drops by vapor diffusion against 20 to 25% (NH4)2SO4, in 100 mM-sodium acetate buffer, pH 5.0. The crystals diffract in the X-ray beam to 2.8 A resolution. Their space group is P2(1)2(1)2(1) with cell dimensions a = 81 A, b = 88 A and c = 135 A. Assuming one monomer (Mr 70,362) per asymmetric unit, the solvent content of these crystals is 63%.     

Crystallization and preliminary analysis of enzyme-substrate complexes of pyruvate kinase from rabbit muscle.

Pyruvate kinase from rabbit muscle has been crystallized in a form suitable for high resolution X-ray analysis. Complexes of the enzyme with Mn2+ and either pyruvate or oxalate crystallize from solutions of polyethyl-eneglycol 8000 at pH 6.0. Crystals obtained from solutions of the complexes with pyruvate or oxalate appear isomorphous and belong to the triclinic space group P1. The crystals have unit cell dimensions a = 83.3(4) A, b = 109.4(6) A, c = 145.7 (7) A, alpha = 94.9 degrees, beta = 93.6 degrees, gamma = 112.2 degrees. These crystals diffract to better than 2.4 A resolution and are stable in the X-ray beam for at least 20 hr. Electron paramagnetic resonance measurements on a single crystal show that Mn2+ is bound to the crystalline protein.