Crystallization and preliminary X-ray diffraction studies on a soluble Escherichia coli quinone oxidoreductase

Single crystals suitable for X-ray diffraction studies have been obtained from a soluble Escherichia coli NAD(P)H-dependent quinone oxidoreductase (QOR) of molecular mass 35 kDa. Crystals were grown by the vapour diffusion method in the presence of NADPH and diffract strongly to beyond 2.0 A. The space group was determined to be P2(1)2(1)2(1) with unit cell dimensions of a = 107.44 A, b = 104.06 A, c = 77.45 A. There is one dimer in the asymmetric unit.     

Purification, characterization, crystallization, and preliminary X-ray results from Paracoccus denitrificans porin

The porin from Paracoccus denitrificans ATCC 13543 was purified and crystallized. Two crystal forms were obtained from porin solutions with beta-d-octylglucopyranoside as detergent. Crystals of form I belong to the monoclinic spacegroup C2 with unit cell dimensions a = 112.2 A, b = 193.8 A, c = 100.5 A and beta = 129.2 degrees. There is 1 trimer per asymmetric unit. Crystals of form II are triclinic with a = 89.7 A, b = 98.8 A, c = 112.5 A, alpha = 112.5 degrees, beta = 101.8 degrees, gamma = 106.7 degrees (2 trimers per asymmetric unit). Both crystal forms diffract to 3 A.     

Angina pectoris: progress in diagnosis and treatment. I. Physiopathology. 2. Symptoms most likely associated with angina pectoris]

Two isozymes of 6-phosphoglucose isomerase (phosphoglucose isomerase A & phosphoglucose isomerase B), isolated from Bacillus stearothermophilus, have been overexpressed in Escherichia coli strain DF2145 and purified to homogeneity. Crystals of both isozymes have been obtained by the vapor diffusion method. The crystals of phosphoglucose isomerase A have unit cell dimensions a = b = 132.0 A, c = 183.6 A, and diffract to about 2.8 A resolution. An analysis of the reflection data indicates that the crystal system is hexagonal, space group P6122 or P6522. The crystals of phosphoglucose isomerase B complexed with 6-phosphogluconate belong to the orthorhombic space group I222 (or I212121), with cell dimensions a = 75.1 A, b = 95.7 A, c = 171.5 A, and diffract to a resolution of 2.3 A. These crystals promise to yield more detail for the substrate recognition and higher resolution structures of 6-phosphoglucose isomerase.     

Crystallization and atomic resolution X-ray diffraction analysis of antichymotrypsin variants

Crystals of two recombinant antichymotrypsin (rACT) variants have been prepared: variant rACT-T345R crystallizes in space group P2(1) (a = 109.2 A, b = 79.4 A, c = 111.9 A, beta = 116.3 degrees, with 2 molecules in the asymmetric unit), and variant ACT' crystallizes in space group P2(1)22(1) (a = 69.7 A, b = 77.2 A, c = 83.8 A, with one molecule in the asymmetric unit). The latter variant is an engineered dimer having the P3-P3' hexapeptide sequence of the related serpin, alpha 1-proteinase inhibitor, substituted for the corresponding wild-type sequence. Crystals of each variant diffract to a limiting resolution of 2.5 A, which represents the best diffraction yet achieved for a crystalline, inhibitory serpin. The exceptional quality of ACT' crystals probably arises from favorable protein-protein interactions as well as a stabilizing disulfide crosslink engineered between the monomers.     

Crystallization and preliminary crystallographic studies of Gi alpha 1 and mutants of Gi alpha 1 in the GTP and GDP-bound states

Several different crystal forms of Gi alpha 1 have been grown and analyzed. Crystals of native protein containing bound GTP gamma S belong to space group P3(1)2(1) or P3(2)2(1) with cell dimensions a,b = 80.6 A and c = 106.3 A and diffract to a resolution of 1.9 A using synchrotron radiation. Crystals of native protein containing bound GDP belong to space group I4 with cell dimensions a,b = 121.3 A, and c = 67.7 A and diffract to 3.0 A. Data sets from crystals grown using mutant proteins have also been obtained and characterized.     

Crystallization and preliminary crystallographic data of the alpha-amylase inhibitors, Haim I and Paim I

Two proteins that act as alpha-amylase inhibitors, Haim I and Paim I, were crystallized and preliminary X-ray diffraction studies on them were carried out. We also sequenced Haim I prepared from Streptomyces griseosporeus YM-25 and confirmed that it is composed of 78 amino acid residues. Crystals of Haim I were grown from ammonium sulfate solution mixed with ethanol by the vapor diffusion technique. The crystals grew as hexagonal bipyramids and diffracted X-rays beyond 2.0 A resolution. They belong to the space group P6(1)22 (or P6(5)22) with unit cell dimensions of a = b = 36.7 A, c = 192.4 A, and contain one molecule per asymmetric unit. Paim I, a protein of 39 amino acid residues produced by Streptomyces corchorusii, was crystallized under similar conditions to Haim I. The crystals diffracted X-rays beyond 2.5 A. They belong to the space group P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = b = 65.4 A, c = 96.1 A, and contain three molecules per asymmetric unit.     

Crystallization and preliminary crystallographic analysis of an amylopullulanase from the hyperthermophilic archaeon Pyrococcus woesei

The thermostable amylopullulanase from Pyrococcus woesei was crystallized. Crystals, suitable for a crystallographic analysis up to a size of 0.6 mm in their longest dimension, have been obtained by the vapor diffusion method in a solution containing polyethyleneglycol 4000 (PEG 4000), isopropanol, and Tris/Cl- buffer pH 7.5. Crystals grown under these conditions form hexagonal rods and diffract to a maximum resolution of 3 A. The crystals belong to the trigonal lattice type with the spacegroup P3(1)21 or P3(2)21, respectively, have the cell dimensions a = b = 96.8 A, c = 196.2 A, alpha = beta = 90 degrees, gamma = 120 degrees. The crystals have a theoretical packing density of 2.7 A3/Da, assuming one molecule with a molecule weight of 88.8 kDa in the asymmetric unit. Furthermore the self-rotation analysis of the dataset revealed only crystallographic symmetries. The merged native data of two crystals resulted in a 88% complete dataset.     

Purification and crystallization of benzoylformate decarboxylase

A new large-scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X-ray crystallographic study of the enzyme. The previously observed instability of the enzyme was overcome by addition of 100 microM thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57-kDa subunits. Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 A resolution and are stable for days to X-ray radiation. Analysis of X-ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as I222. The unit cell dimensions are a = 82 A, b = 97 A, c = 138 A. An average Vm value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged with tetrahedral 222 symmetry.     

Bacteriophage T7 DNA ligase. Overexpression, purification, crystallization, and characterization

The bacteriophage T7 DNA ligase gene was amplified using polymerase chain reaction-based methods and cloned into a T7 promoter-based expression vector. The protein was overexpressed to greater than 15% of total soluble protein and purified to homogeneity, yielding 60-70 mg of protein per liter of bacterial culture. An initial physical and biochemical characterization of the enzyme reveals that it exists as a monomer and can ligate nicked, cohesive, and blunt-ended DNA fragments. Inhibition of the enzyme activity by a nonhydrolyzable ATP analogue was also investigated. The enzyme has been crystallized from methoxypolyethylene glycol. The crystals are of the orthorhombic space group P2(1)2(1)2 and diffract to 2.6 A. The unit cell dimensions are a = 66.1 A, b = 87.6 A, and c = 78.6 A, with one monomer in the asymmetric unit (Vm = 2.77 A3/Da). This is the first member of the DNA ligase family of enzymes to be crystallized.     

Crystallization and preliminary X-ray studies on pseudoazurin from Achromobacter cycloclastes IAM1013

New crystals of a blue copper protein, pseudoazurin from denitrifier Achromobacter cycloclastes IAM1013, have been obtained by means of vapor diffusion with ammonium sulfate as a precipitant at pH 6.0 and 4 degrees C. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 56.69(2), b = 61.53(2), and c = 30.20(1) A. The asymmetric unit includes one molecule of pseudoazurin with a Vm value of 2.04 A3/Da. The crystals are so stable against X-ray irradiation that a complete data set up to 1.54 A has been collected using a single native crystal. Solution of the structure was performed by means of the Patterson search techniques, and the current crystallographic R-factor is 17.5% at 3.0 A resolution. Refinement at higher resolution is in progress.     

Subunit composition, crystallization and preliminary crystallographic studies of the Desulfovibrio gigas aldehyde oxidoreductase containing molybdenum and [2Fe-2S] centers

The Desulfovibrio gigas aldehyde oxidoreductase contains molybdenum bound to a pterin cofactor and [2Fe-2S] centers. The enzyme was characterized by SDS/PAGE, gel-filtration and analytical ultracentrifugation experiments. It was crystallized at 4 degrees C, pH 7.2, using isopropanol and MgCl2 as precipitants. The crystals diffract beyond 0.3-nm (3.0-A) resolution and belong to space group P6(1)22 or its enantiomorph, with cell dimensions a = b = 14.45 nm and c = 16.32 nm. There is one subunit/asymmetric unit which gives a packing density of 2.5 x 10(-3) nm3/Da (2.5 A3/Da), consistent with the experimental crystal density, rho = 1.14 g/cm3. One dimer (approximately 2 x 100 kDa) is located on a crystallographic twofold axis.     

A new crystal form of proteinase A, a non-pepsin-type acid proteinase from Aspergillus niger var. macrosporus

Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase, whose catalytic residues and mechanism remain to be elucidated. A new form of proteinase A crystals more suitable for crystallography than that obtained previously was prepared from an ammonium sulfate solution at pH 3.5 by the hanging-drop vapor diffusion method. The space group of the crystals was P2(1)2(1)2(1), with unit cell dimensions of a = 69.75 +/- 0.06 A, b = 87.55 +/- 0.05 A, and c = 60.83 +/- 0.04 A. On the assumption of two enzyme molecules per asymmetric unit, the calculated volume to unit protein mass ratio (Vm) was 2.08 A3/Da. By assuming the specific volume to be 0.74 cm3/g, the solvent content (Vso1) was estimated to be 41%, i.e., much larger than that of the crystal form obtained previously at pH 2.0 (Vso1 = 26%). Diffraction data were collected up to a resolution higher than 1.6 A, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation.     

Crystallization and preliminary X-ray investigation of calmodulin

Crystals of rat testis calmodulin, a multifunctional Ca2+-binding protein have been grown from solutions of 2-methyl-2,4-pentanediol. The crystals are triclinic, space group P1, with a = 29.79(4) A, b = 53.74(7) A, c = 24.78(3) A, alpha = 93.46(2)degrees, beta = 96.98(2)degrees, and gamma = 89.05(3)degrees. There is 1 calmodulin molecule per unit cell. The crystals are quite stable to x-rays and diffract beyond 2.5 A resolution.     

A study of penetrance of Drosophila melanogaster forked gene

Group I intron endonuclease I-CreI is encoded by an open reading frame contained within a self-splicing intron in the Chlamydomonas reinhardtii chloroplast 23S rRNA gene. I-CreI initiates the lateral transfer or homing of this intron by specifically recognizing and cleaving a pseudopalindromic 19-24 bp homing site in chloroplast 23S rRNA genes that lack the intron. The gene encoding this enzyme has been subcloned, and the protein product has been purified and crystallized. The crystals belong to space group P321, with unit cell dimensions a = b = 78.2 A, c = 67.4 A. The crystal unit cell is consistent with an asymmetric unit consisting of the enzyme monomer. The specific volume of this unit cell is 3.3 A3/Da. The crystals diffract to at least 3.0 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector.     

Crystallization and preliminary X-ray diffraction analysis of gingipain R2 from Porphyromonas gingivalis in complex with H-D-Phe-Phe-Arg-chloromethylketone

Gingipain R2 is a 50 kDa proteinase from the oral pathogenic bacterium Porphyromonas gingivalis. This proteinase, which displays no significant sequence homology to any protein previously analyzed by X-ray crystallography, has been crystallized using the vapor diffusion method. Two different crystal forms were obtained from a solution containing polyethylene glycol (MW 8,000) (space group P2(1)2(1)2(1)) or magnesium sulfate (space group R3) as precipitating agent. Complete diffraction data sets have been collected up to 2.0 and 2.9 A resolution, respectively. Cell dimensions are a = 51.9 A, b = 79.9 A, and c = 99.6 A (P2(1)2(1)2(1)), and a = b = 176.6 A, and c = 143.4 A (R3). Considerations of the possible values of Vm accounts for the presence of one monomer per asymmetric unit in the case of the orthorhombic crystal form, whereas the rhombohedral crystal form, together with the analysis of the self-rotation function, could accommodate a tetramer in the asymmetric unit.     

Anaerobic crystallisation of an isopenicillin N synthase.Fe(II).substrate complex demonstrated by X-ray studies

Isopenicillin N synthase (IPNS) was cocrystallised with ferrous sulphate and its substrate, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (Aad-Cys-Val). Vital to the successful procedure was the maintenance of a rigorously anaerobic environment. Hanging-drop vapour-diffusion crystallisation experiments, using lithium sulphate as the precipitant produced three crystal forms. Form I crystals, with a plate habit, diffracted X-rays to at least 0.11-nm resolution at the European Synchrotron Radiation Facility and belong to the space group P2(1)2(1)2(1), with unit-cell dimensions a = 4.68, b = 7.15, c = 10.10 nm. Their asymmetric unit contains a single IPNS.Fe(II).Aad-Cys-Val complex with a solvent content of 38.5%. Form II crystals, with a hexagonal habit, diffract X-rays to at least 0.21 nm resolution at the European Synchrotron Radiation Facility and belong to the space group P3(1)21, with unit-cell dimensions a = 10.10, b = 10.10, c = 11.567 nm. Their asymmetric unit also contains a single IPNS.Fe(II).Aad-Cys-Val complex with a solvent content of 69.5%. Form III crystals, needles, do not show well-ordered diffraction. Although all three forms were initially produced in crystallisation experiments under identical conditions, appropriate micro and streak seeding allows selective crystallisation of form I or form II crystals. Extended X-ray-absorption fine-structure studies on a crystalline slurry of the form I crystals demonstrate the presence of an Fe-S(Aad-Cys-Val) bond length of 0.234 +/- 0.003 nm.     

Crystallization and preliminary diffraction analysis of a truncated homodimer of human phenylalanine hydroxylase

A recombinant truncated form (delta1-102/delta428-452) of the non-heme iron-dependent metalloenzyme human phenylalanine hydroxylase (hPAH, phenylalanine 4-monooxygenase; EC 1.14.16.1) was expressed in E. coli, purified to homogeneity as a homodimer (70 kDa) and crystallized using the hanging drop vapour diffusion method. The crystals are orthorhombic, space group C222 with cell dimensions of a = 66.6 A, b = 108.4 A, c = 125.7 A. The calculated packing parameter (Vm) is 3.24 A3/Da with four 2-fold symmetric dimers (or eight momomers) in the unit cell. Data have been collected to 2.0 A resolution.     

Crystallization and preliminary crystallographic analysis of RepA1, a replication control protein of the RepFIC replicon of enterotoxin plasmid EntP307

RepA1 protein is essential for replication of the RepFIC replicon of enterotoxin plasmid EntP307 and is thought to interact directly with the origin of replication. We have purified RepA1 from an over-producing expression system and have prepared single crystals using a macroseeding technique. The crystals belong to space group P2(1)2(1)2(1) or P2(1)2(1)2, with cell dimensions a = 61 A, b = 67 A, and c = 243 A. They diffract X-rays to 3.3 A resolution and probably contain two 40,000 molecular weight RepA1 molecules per asymmetric unit.     

Scavenger receptors may regulate nitric oxide production from macrophages stimulated by LPS

A new crystal form of papain from the latex of Carica papaya, complexed with an inhibitor (Z-Arg-Leu-Val-Gly-CHN2) was obtained by the vapor-diffusion method using a methanol/ethanol mixture as a precipitant. The slat-like crystals are monoclinic, space group P2(1), with unit cell parameters a = 42.6 A, b = 49.8 A, c = 50.5 A, A = 111.9 degrees, and contain one molecule in the asymmetric unit. The crystals are stable in the X-ray beam and diffract beyond 1.8 A. A molecular model has been placed in the unit cell by molecular replacement.     

Preliminary crystallographic study of D(-)-mandelate dehydrogenase from Rhodotorula graminis

NAD+ dependent D(-)-mandelate dehydrogenase from the yeast Rhodotorula graminis strain KGX 39 has been crystallized in three different forms using the hanging drop vapour diffusion method at 15 to 20 degrees C. Type I crystals belong to space group P222(1), P22(1)2(1) or P2(1)2(1)2(1) with a = 100.3 A, b = 117.4 A, c = 80.4 A and are likely to contain a dimer in the crystallographic asymmetric unit. They diffract to dmin = 3.0 A. Type II crystals belong to space group P22(1)2(1) or P2(1)2(1)2(1) with a = 187.8 A, b = 122.9 A, c = 72.1 A and contain probably two dimers in the crystallographic asymmetric unit. They diffract to dmin = 1.8 A. Type III crystals belong to space group P2(1)2(1)2(1) with a = 109.6, b = 52.0 A, c = 145.7 A, and are likely to contain a dimer in the crystallographic asymmetric unit. They diffract at least to dmin = 2.5 A.