Crystallization and preliminary X-ray diffraction analysis of catalase HPII from Escherichia coli

Green crystals of the hexameric catalase HPII from Escherichia coli have been obtained by the hanging-drop method. The crystals belong to the monoclinic space group P2 with a = 123 A, b = 132 A, c = 93 A, beta = 112.5 degrees. There are three subunits in the asymmetric unit. The crystals diffract at least to 3.2 A resolution and are suitable for further X-ray diffraction studies.     

Preliminary X-ray studies of the tetra-heme cytochrome c3 and the octa-heme cytochrome c3 from Desulfovibrio gigas

A tetra-heme and an octa-heme cytochrome c3 from the sulfate bacterium Desulfovibrio gigas have been crystallized. Diffraction quality crystals of the tetra-heme cytochrome are obtained from solution by the addition of polyethylene glycol at pH 6.5. The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell parameters a = 42.27 A, b = 52.54 A and c = 52.83 A. The octa-heme cytochrome crystals develop from low ionic strength solutions of phosphate or Tris-Cl in the pH range 6.2-7.6. The crystals belong to the trigonal system, space group P3(1) or the enantiomorph P3(2), with unit cell parameters a = b = 57.4 A, c = 97.3 A, gamma = 120 degrees. Single crystal diffraction studies of the structures of these two low-potential cytochromes are in progress.     

单斜Al－（L－丙氨酸）胰岛素初步晶体学研究

用微量气相扩散方法在含酚的柠檬酸缓冲体系中,得到了可供Ｘ射线晶体学分析用的Ａｌ－（Ｌ－丙氨酸）胰岛素晶体,晶体衍射能力为２．５Ａ。经Ｘ射线衍射分析确定,该晶体属于单斜晶系,空间群为Ｐ２_１,晶胞参数：ａ＝６１．５Ａ,ｂ＝６２．２Ａ,ｃ＝４８．３Ａ,α＝γ＝９０．０°,β＝１１０．９°。晶胞中每个结晶学不对称单位含有一个Ａｌ－（Ｌ－丙氨酸）胰岛素六聚体。     

Preliminary crystallographic studies of urease from jack bean and from Klebsiella aerogenes.

Ureases from both jack bean (Canavalia ensiformis) seeds and Klebsiella aerogenes have been crystallized by the hanging drop method. The plant-derived urease crystals are regular octahedra analogous to those obtained by Sumner. Preliminary X-ray diffraction studies show that the crystals belong to the cubic space group F4(1)32, with a = 364 A, and appear to contain one or two subunits in the asymmetric unit. Using a synchrotron source, the crystals diffract to near 3.5 A resolution. Crystals of urease from K. aerogenes belong to the cubic space group I23 or I2(1)3, with a = 170.8 A and appear to contain a single catalytic unit per asymmetric unit. The crystals diffract to better than 2.0 A resolution and are well suited for structural analysis.     

Crystallization and preliminary X-ray studies on human epidermal growth factor

Single crystals of human epidermal growth factor have been prepared from a polyethylene glycol solution and characterized by X-ray diffraction. The crystals grow in a space group of P2(1) with unit cell dimensions of a = 32.7, b = 32.5, c = 22.3 A, and beta = 96.9 degrees. They contain a single molecule per asymmetric unit and show better diffraction than 2.5 A.     

X-ray analysis of new crystal forms of the sweet protein thaumatin

Thaumatin is a plant protein that in the mature form contains 8 disulfide bonds and 207 amino acids. Several forms of this protein occur naturally and each elicits an intense sweetness sensation when tasted in microgram quantities. The two major forms of thaumatin are easily separable by ion exchange chromatography. Crystals of the two proteins (designated here A and B) have been grown by vapor equilibration from solutions containing polyethylene glycol and examined by X-ray diffraction. The thaumatin A crystals are of space group P2(1)2(1)2(1) with a = 44.3 A, b = 63.7 A and c = 72.7 A. The crystals of thaumatin B are of space group C2 with a = 117.7 A, b = 44.9 A, and c = 38.0 A and beta = 94.0 degrees. Both crystals diffract to well beyond 2.3 A and appear suitable for high resolution structure analysis. Four heavy atom derivatives of thaumatin B have been generated and diffraction data to 4 A resolution have been collected. This work is designed to provide a basis for studying the 3-dimensional structure of more than 100 genetically generated thaumatin derivatives, several of which show enhanced stability and improved taste characteristics.     

Characterization of crystals of an intact monoclonal antibody for canine lymphoma

A monoclonal antibody of the subclass IgG2a specific for canine lymphoma cells has been crystallized by vapor diffusion from polyethylene glycol 8000. the crystals, which occasionally measure nearly a millimeter on edge, have been examined by X-ray diffraction. The crystals are of triclinic space group P1 with unit cell parameters of a = 66.39 A, b = 77.34 A, c = 101.42 A, alpha = 87.60 degrees, beta = 92.55 degrees, gamma = 97.54 degrees and cell volume of V = 4.84 x 10(5) A3. There is one entire antibody molecule as the asymmetric unit of the crystals. Three-dimensional X-ray diffraction data have been collected to 2.8 A resolution and a self rotation function calculation shows a pronounced peak indicating at least an approximate non-crystallographic dyad axis.     

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 allosteric L-lactate dehydrogenase from Thermus caldophilus and preliminary crystallographic data

L-Lactate dehydrogenase of Thermus caldophilus GK24 was purified from Escherichia coli containing an overexpression plasmid. The enzyme was crystallized from polyethylene glycol 6000 solutions without ligands by the hanging drop vapor diffusion method. Two forms of crystals were obtained. The crystals grown at pH 6.0 were characterized by means of an X-ray diffraction experiment, while those grown at pH 6.5 and 7.0 did not give detectable diffraction spots. The crystals grown at pH 6.0 belonged to monoclinic space group P2(1), the cell dimensions being a = 54.8 A, b = 138.2A, c = 86.1 A, and beta = 93.3 degrees. These crystals diffract to beyond 2.5 A spacing and are stable on X-ray irradiation.     

Preliminary investigation of crystals of the neutral lipase from Pseudomonas fluorescens

The neutral lipase from the bacteria Pseudomonas fluorescens, marketed under the trade name LpL-200S, has been crystallized in a form suitable for X-ray diffraction analysis from 35% n-propanol at pH 8.5. The crystals are monoclinic prisms and are of space group C2 with a = 91.00 A, b = 47.17 A, c = 35.21 A and beta = 121.43 degrees. There is one molecule of the protein as the asymmetric unit of the crystals. The diffraction pattern extends to at least 1.6 A resolution and the crystals are extremely robust in terms of X-ray exposure.     

Crystallization and preliminary X-ray data of a phleomycin-binding protein from Streptoalloteichus hindustanus

Large single crystals of a glycopeptide resistance protein encoded by the SH-ble gene from Streptoalloteichus hindustanus have been grown using vapor diffusion techniques with ammonium sulfate as the precipitant. The diffraction pattern extends to 2.0 A resolution. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2 and have unit cell dimensions of a = 48.8 A and c = 112.5 A.     

Crystallographic characterization of recombinant human interleukin 2

Recombinant human interleukin 2 produced by Escherichia coli was purified to homogeneity and crystallized after being separated from methionyl interleukin 2. Crystals suitable for structural studies have been obtained by the seed enlargement technique, using the method of vapor diffusion with ammonium sulfate as the precipitant at pH 4.6. The space group is P2(1)2(1)2 with cell dimensions a = 49.2 A, b = 87.6 A and c = 32.4 A. The asymmetric unit contains one molecule of the protein. From preliminary results, the crystals are moderately stable to X-rays and produce measurable reflections to a resolution of about 2.2 A. The diffraction data for the native crystals have been collected on a diffractometer at 2.4 A resolution.     

Crystallization of GST2, a human class alpha glutathione transferase

Single crystals of human GST2, a class alpha glutathione transferase have been grown in polyethylene glycol 2000 by the hanging-drop vapour diffusion method. The crystals belong to space group C2 and have cell dimensions a = 100.8 A, b = 95.4 A, c = 105.2 A and beta = 92.4 degrees. The X-ray diffraction pattern extends to better than 3 A resolution.     

Crystallization and preliminary x-ray characterization of thioredoxin reductase from Escherichia coli

Single crystals of thioredoxin reductase, suitable for x-ray diffraction studies, have been obtained at room temperature by vapor diffusion of 10-20 mg/ml protein solution against 35% polyethylene glycol containing 200 mM ammonium sulfate. Good quality crystals appear spontaneously only from a protein solution that had been stored for more than a year at 4 degrees C, although large single crystals are reproducibly obtained from fresh protein solutions by micro-seeding. The space group is P6(3)22 (a = b = 123.8 A, c = 81.6 A), with one monomer of the enzyme (34.5 kDa) in the crystallographic asymmetric unit. The crystals are well ordered and diffract to beyond 2 A resolution.     

Purification, crystallization and preliminary X-ray data of the bacteriophage MS2

Single crystals of the bacteriophage MS2 have been produced by the vapour diffusion technique in the presence of 1.5% polyethylene glycol 6000 and 0.2 M-sodium phosphate buffer (pH 7.4). These are the first bacteriovirus crystals diffracting to high resolution. The crystal space group is C2 with the unit cell parameters a = 467.9 A, b = 289.5 A, c = 275.6 A and beta = 121.8 degrees. The asymmetric unit contains one half of the virion. The maximum resolution limit of the X-ray diffraction data obtained from these crystals was 2.9 A. The purification of the virus material was done by mild procedures exclusively and involved precipitation with polyethylene glycol 6000 and size exclusion chromatography on Sepharose CL-4B.     

Crystallization and preliminary crystallographic study of bacterial alpha-amylases

Crystallization of bacterial alpha-amylases has been achieved by the hanging-drop vapor diffusion method. The crystals of Bacillus licheniformis and B. licheniformis 584 amylases are isomorphous to each other. The crystals of B. licheniformis amylase belong to the tetragonal system, space group P4(2)2(1)2 with cell dimensions of a = 119.3 and c = 85.4 A. The asymmetric unit contains one molecule of amylase, with a volume per molecular mass, Vm, of 2.75 A3/Da. The crystals of B. licheniformis and B. licheniformis 584 amylases diffract beyond 2.5 A resolution and are suitable for X-ray diffraction analysis. The crystals of B. amyloliquefaciens amylase are orthorhombic, and have space group C222(1), with cell dimensions of a = 154, b = 298, and c = 90 A. The asymmetric unit contains three to five molecules. In the crystallization of B. licheniformis and B. licheniformis 584 amylases, the addition of EDTA was indispensable to obtain large single crystals, while it had an adverse effect on the crystallization of B. amyloliquefaciens amylase, producing a large amount of small crystals.     

Expression, purification and crystallization of the Vibrio harveyi acyltransferase.

We have obtained X-ray quality single crystals of Vibrio harveyi acyltransferase. The protein was obtained from V. harveyi by a gene mobilization expression system. The crystals are monoclinic (space group P2(1), a = 89.9 A, b = 83.6 A, c = 47.1 A, beta = 97.3 degrees) with two molecules related by a pronounced non-crystallographic dyad in the asymmetric unit, with a solvent content of approximately 50%. The diffraction pattern from fresh crystals extends beyond 2 A resolution using sealed tube CuK alpha radiation. The elucidation of the three-dimensional structure of this enzyme, believed to contain a proteinase-like catalytic triad, which resembles in many ways other eukaryotic fatty acid chain terminating enzymes, may have important consequences for our understanding of the molecular basis of the final stages of the synthesis of fatty acids.     

Crystallization of rat intestinal fatty acid binding protein. Preliminary X-ray data obtained from protein expressed in Escherichia coli

Rat intestinal fatty acid binding protein has been expressed in Escherichia coli, purified with bound long chain fatty acids and crystals grown from solutions of polyethylene glycol 4000. The crystals are monoclinic, space group P2(1), a = 3638 A, b = 57.2 A, c = 31.9 A, and beta = 113.9 degrees. Each unit cell contains two monomers of this 132-residue, 15.1-kDa polypeptide. The crystals are remarkably resistant to x-ray damage. X-ray diffraction data have been observed to 2.0 A resolution. Platinum chloride was used to generate a potential isomorphous heavy atom derivative.     

Crystallization and preliminary crystal data of porcine pepsinogen

Single crystals of porcine pepsinogen, suitable for x-ray diffraction studies, have been grown with lithium sulfate as the precipitant. These pepsinogen crystals were dissolved, activated, and assayed for proteolytic activity. The specific enzymic activity of the dissolved crystalline protein was nearly twice that of the commerical pepsinogen from which the crystals were grown. Incubation at pH 8 before assay demonstrated that the crystals are free of pepsin. This crystal form of pepsinogen belongs to the monoclinic space group C2 with 4 molecules in the unit cell. The unit cell dimensions are a = 104.8 +/- 0.5 A, b = 43.1 +/- 0.1 A, c = 88.4 +/- 0.3 A, and beta = 91.3 degrees.     

Crystallization and preliminary data of Indian buffalo erythrocyte carbonic anhydrase

The most abundant anhydrase isoenzyme from the erythrocyte of Indian buffalo has been purified using affinity gel and DEAE-cellulose ion-exchange columns and single crystals suitable for X-ray diffraction studies have been obtained. The unit cell dimensions are a = 46.8 A, b = 104.5 A, c = 60.4 A, beta = 91.2 degrees and the space group is P2(1), with two molecules per asymmetric unit.