Crystallization and preliminary X-ray diffraction studies of a bacterial flavohemoglobin protein

A flavohemoglobin protein (FHP) was isolated from Alcaligenes eutrophus and has been crystallized by vapor diffusion methods using PEG 3350 as precipitant. The crystals of the FAD- and heme-containing protein belong to the monoclinic space group P2₁ with unit cell parameters of 52.2 Å, 85.8 Å, 103.9 Å, and 81.8° corresponding to two molecules per asymmetric unit. The crystals diffract at least to a resolution of 2.0 Å and are suitable for an X-ray structure analysis. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray diffraction studies of mandelonitrile lyase from almonds

Single crystals of three different isoenzymes of (R)?(+) mandelonitrile lyase (hydroxynitrile lyase) from almonds (Prunus amygdalus) have been obtained by hanging drop vapor diffusion using polyethylene glycol 4000 and isopropanol as co-precipitants. The crystals belong to the monoclinic space group P2_l with unit cell parameters a = 69.9, b = 95.1, c = 95.6 Å, and β = 118.5°. A complete set of diffraction data has been collected to 2.6 Å resolution on native crystals of isoenzyme III. © 1994 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray diffraction studies of the cartilage link protein from bovine trachea

Cartilage extracellular matrix link protein, having molecular mass of approximately 40 kDa, is a metalloprotein that binds divalent cations and is only soluble in low ionic strength solutions. The link protein was purified from bovine trachea and has been crystallized by a vapor diffusion method using PEG 3350 as precipitant. The crystal symmetry is P1, and the unit cell dimensions are a = 43.55, b = 53.11, c = 60.10 Å, α = 90.44, β = 106.21, γ = 101.51°. The V_M of 1.8 Å₃/Da is consistent with the presence of two molecules of the link protein in the asymmetric unit. The crystals diffract X-rays from a synchrotron source to 1.7 Å resolution. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic studies of UDP-N-acetylenolpyruvylglucosamine reductase.

The overexpression and purification of the second enzyme in Escherichia coli peptidoglycan biosynthesis, UDP-N-acetylenolpyruvylglucosamine reductase (MurB), provided sufficient protein to undertake crystallization and X-ray crystallographic studies of the enzyme. MurB crystallizes in 14-20% PEG 8000, 100 mM sodium cacodylate, pH 8.0, and 200 mM calcium acetate in the presence of its substrate UDP-N-acetylglucosamine enolpyruvate. Crystals of MurB belong to the tetragonal space group P4(1)2(1)2 with a = b = 49.6 A, c = 263.2 A, and alpha = beta = gamma = 90 degrees at -160 degrees C and diffract to at least 2.5 A. Screening for heavy atom derivatives has yielded a single site that is reactive with both methylmercury nitrate and Thimerosal.     

Crystallization and preliminary X-ray crystallographic studies on Type III antifreeze protein.

Type III antifreeze protein, more specifically the recombinant QAE-Sephadex-binding isoform, has been crystallized in 50-55% saturated ammonium sulfate, 0.1 M sodium acetate, pH 4.0-4.5. The resultant crystals belong to the orthorhombic space group P212121 with a = 32.60 A, b = 39.00 A, and c = 46.57 A and diffract to at least 1.7 A. A set of 1.7-A native data has been collected, with completeness 93.4% and Rsym of 0.069. Initial screening for heavy-atom derivatives has yielded a Pt-bound derivative.     

Crystallization and preliminary X-ray diffraction studies of peroxidase from a fungus Arthromyces ramosus

Peroxidase (donor: H₂O₂ oxi-doreductase [EC 1.11.1.7]) was purified from the culture broth of the hyphomycete Arthromyces ramosus in the early log phase to show a single band on SDS-PAGE. The crystals of A. ramosus peroxidase (ARP) were formed by salting out with ammonium sulfate at room temperature and pH 7.5. The repeated seeding technique was employed to grow the crystals to the size large enough for X-ray diffraction study. The crystals were characterized as tetragonal, space group P4₂2₁2, with unit cell dimensions of a = b = 74.5 Å, c = 117.6 Å. The asymmetric unit contains one molecule of peroxidase. They diffract X-rays to at least 2.0 Å resolution and are stable to X-rays. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray diffraction analysis of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA Synthetase, a class I aminoacyl tRNA synthetase playing a crucial role in protein biosynthesis, has been crystallized for the first time. Polyethylene glycol (PEG) was used as a precipitant, and the crystallization proceeded at pH 6.5. These single crystals diffracted to 2.8 A with a rotating anode X-ray source and R-axis IIc image plate detector. They have an orthorhombic space group P2(1)2(1)2 with unit cell parameters of a = 251.51 A, b = 53.12 A, and c = 52.35 A. A complete native data set has been collected at 3.1 A resolution for these crystals.     

Crystallization and preliminary X-ray analysis of membrane-bound pyrophosphatases

Abstract

Membrane-bound pyrophosphatases (M-PPases) are enzymes that enhance the survival of plants, protozoans and prokaryotes in energy constraining stress conditions. These proteins use pyrophosphate, a waste product of cellular metabolism, as an energy source for sodium or proton pumping. To study the structure and function of these enzymes we have crystallized two membrane-bound pyrophosphatases recombinantly produced in Saccharomyces cerevisae: the sodium pumping enzyme of Thermotoga maritima (TmPPase) and the proton pumping enzyme of Pyrobaculum aerophilum (PaPPase). Extensive crystal optimization has allowed us to grow crystals of TmPPase that diffract to a resolution of 2.6 Å. The decisive step in this optimization was in-column detergent exchange during the two-step purification procedure. Dodecyl maltoside was used for high temperature solubilization of TmPPase and then exchanged to a series of different detergents. After extensive screening, the new detergent, octyl glucose neopentyl glycol, was found to be the optimal for TmPPase but not PaPPase.     

Crystallization and preliminary X-ray analysis of Escherichia coli methionyl–tRNA formyltransferase

Methionyl–tRNA formyltransferase from Escherichia coli, a monomer of 34kDa, was overexpressed from its cloned gene fmt (Guillon, J.M., Mechulam, Y., Schmitter, J.M., Blanquet, S., and Fayat, G., J. Bacteriol. 174:4294–4301, 1992) and crystallized using ammonium sulphate as precipitant. The crystals are trigonal and have unit cell parameters a = b = 151.0Å, c = 81.8Å. They belong to space group P3₂21 and diffract to 2.0Å resolution. The structure is being solved by multiple isomorphous replacement. © 1996 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of phosphoribulokinase from Rhodobacter sphaeroides.

A recombinant form of Rhodobacter sphaeroides phosphoribulokinase (PRK), expressed in Escherichia coli and isolated by affinity chromatography, was crystallized by the sitting drop vapor diffusion technique using NH4H2PO4 (pH 5.6) as the precipitating agent. PRK crystallizes in the cubic space group P432, with unit cell parameters a = b = c = 129.55 A. Based on the assumption of one 32-kDa monomer per asymmetric unit, the Vm value is 2.83 A3/Da. The octameric molecular symmetry is consistent with two planar tetramers stacked in a nearly eclipsed arrangement. A native data set has been collected to 2.6 A resolution.     

Crystallization and preliminary diffraction studies of thioesterase II from rat mammary gland

Thioesterase II from rat mammary gland has been crystallized in the presence of decanoic acid by the vapor diffusion method. The crystals belong to the orthorhombic space group P2₁2₁2₁, and have cell dimensions, a = 52.7 Å, b = 78.0 Å, and c = 133.6 Å. The asymmetric unit likely consists of two protein monomers based on predictions from its calculated Matthews coefficient. Crystals typically diffract to at least 2.5 Å resolution and are suitable for X-ray crystallographic analysis. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray diffraction studies of the human adenovirus serotype 2 proteinase with peptide cofactor.

Recombinant human adenovirus serotype 2 proteinase (both native and selenomethionine-substituted) has been crystallized in the presence of the serotype 12, 11-residue peptide cofactor. The crystals (space group P3(1)21 or P3(2)21, one molecule per asymmetric unit, a = b = 41.3 angstrum, c = 197.0 angstrum) grew in solutions containing 20-40% 2-methyl-2,4-pentanediol (MPD), 0.1-0.2 M sodium citrate, and 0.1 M sodium HEPES, pH 5.0-7.5. Diffraction data (84% complete to 2.2 angstrum resolution with Rmerge of 0.0335) have been measured from cryopreserved native enzyme crystals with the Argonne blue (1,024 x 1,024 pixel array) charge-coupled device detector at beamline X8C at the National Synchrotron Light Source (operated by Argonne National Laboratory's Structural Biology Center). Additionally, diffraction data from selenomethionine-substituted proteinase, 65% complete to 2.0 angstrum resolution with Rmerge values ranging 0.05-0.07, have been collected at three X-ray energies at and near the selenium absorption edge. We have determined three of the six selenium sites and are initiating a structure solution by the method of multiwavelength anomalous diffraction phasing.     

Crystallization and preliminary X-ray diffraction studies of the human major histocompatibility antigen HLA-B27.

The class I major histocompatibility (MHC) antigen HLA-B27 was purified by immunoaffinity chromatography from the homozygous human B lymphoblastoid cell line LG-2. Detergent-soluble HLA-B27 was cleaved with the protease papain to remove the hydrophobic transmembrane region and the cytoplasmic tail. Crystals of the resulting water-soluble extracellular fragments were obtained in hanging drops by the vapor-diffusion method. The crystals are triclinic, space group P1, with unit cell dimensions a = 45.9 A, b = 71.0 A, c = 83.7 A, alpha = 79.4 degrees, beta = 88.5 degrees, gamma = 89.9 degrees, and diffract beyond 2.5 A resolution.     

Crystallization and preliminary X-ray diffraction analysis of recombinant pentalenene synthase.

Recombinant pentalenene synthase, a 42.5-kDa sesquiterpene cyclase originally isolated from Streptomyces UC5319 and cloned in Escherichia coli, has been crystallized in space group P6(3) with unit cell dimensions a = b = 183.5 A and c = 56.5 A. Hexagonal prismatic crystals, approximately 0.2 x 0.2 x 0.3 mm, diffract to approximately 2.9 A resolution using monochromatic synchrotron radiation. From the universal (and achiral) building block, farnesyl pyrophosphate, pentalenene synthase catalyzes the formation of four stereocenters in the construction of the three fused five-membered rings of pentalenene; this novel sesquiterpene is a precursor to the pentalenolactone family of antibiotics.     

Crystallization and preliminary X-ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus.

Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4 in 50 mM piperazine HCl buffer. The crystals belong to space group P6(2)22 (P6(4)22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6(1)) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one-quarter of a tetramer (M(r) 135,000) in the asymmetric unit and have VM values of 3.8 and 3.3 A3/dalton, respectively). The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A.     

Crystallization and preliminary X-ray diffraction data of two heparin-binding fragments of human fibronectin

Two different heparin-binding fragments of human fibronectin have been crystallized in forms which are suitable for crystal structure analyses. The 30 kDa hep-2A fragment, consisting of type III domains 12–14, was crystallized from solutions containing ammonium sulfate or polyethylene glycol 6000. The crystals grown in ammonium sulfate solutions were orthorhombic with space group I222 or I2₁2₁2₁ with a = 68.1 Å, b = 88.6 Å, and c = 144.9 Å. The crystals grown in polyethylene glycol solutions are hexagonal with space group P6₁22 or P6₅22 witha a = b = 66.7 Å and c = 245.7 Å. The 40 kDa hep-2B fragment, consisting of type III domains 12–15, was also crystallized from solutions containing ammonium sulfate with the addition of glycerol. Glycerol proved an effective agent for reducing the number of crystals in the crystallization experiments, and thus, increasing the size of the crystals in these experiments. This crystal form is nearly isomorphous to the orthorhombic form of the hep-2A fragment with space group I222 or I2₁2₁2₁ and a = 67.5 Å, b = 87.0 Å, and c = 144.3 Å. All crystal forms diffract to at least 3.5 Å resolution and contain a single molecule in the asymmetric unit. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray diffraction studies of leishmanolysin,the major surface metalloproteinase from Leishmania major

The membrane-bound GPI-anchored zinc metalloproteinase leishmanolysin purified from Leishmania major promastigotes has been crystallized in its mature form. Two crystal forms of leishmanolysin have been grown by the vapor diffusion method using 2-methyl-2,4-pentanediol as the precipitant. Macroseeding techniques were employed to produce large single crystals. Protein microhet-erogeneity in molecular size and charge was incorporated into both crystal forms. The tetragonal crystal form belongs to the space group P4₁2₁2 or the enantiomorph P4₃2₁2, has unit cell parameters of a = b = 63.6 Å, c = 251.4 Å, and contains one molecule per asymmetric unit. The second crystal form is monoclinic, space group C2, with unit cell dimensions a = 107.2 Å, b = 90.6 Å, c = 70.6 Å, β = 110.6°, and also contains one molecule per asymmetric unit. Both crystal forms diffract X-rays beyond 2.6 Å resolution and are suitable for X-ray analysis. Native diffraction data sets have been collected and the structure determination of leishmanolysin using a combination of the isomorphous replacement and the molecular replacement methods is in progress. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of squalene-hopene cyclase from Alicyclobacillus acidocaldarius.

The membrane-associated protein squalene-hopene cyclase from Alicyclobacillus acidocaldarius was overexposed in Escherichia coli and purified by ion exchange and gel permeation chromatography. Crystals of three interrelated forms were grown by vapor diffusion under identical conditions. The crystals diffract to about 2.3 A resolution, but they are unstable in the X-ray beam. An interpretable heavy-atom derivative was obtained.     

Crystallization and preliminary X-ray diffraction studies on recombinant isopenicillin N synthase from Aspergillus nidulans.

Recombinant Aspergillus nidulans isopenicillin N synthase was purified from an Escherichia coli expression system. The apoenzyme in the presence of saturating concentrations of MnCl2 could be crystallized by either macro- or microseeding, using the hanging drop vapor diffusion technique with polyethylene glycol 8000 as precipitant. The crystals (0.5-1.0 mm overall dimensions) diffract X-rays to at least 2.0 A resolution at synchrotrons and belong to space group P212121 with unit cell dimensions of a = 59.2 A, b = 127.0 A, and c = 139.6 A. The asymmetric unit contains one dimer, and the solvent content of the crystals is 60%. The crystals are radiation sensitive.     

Crystallization and preliminary X-ray analysis of glucose-fructose oxidoreductase from Zymomonas mobilis.

Glucose-fructose oxidoreductase (E.C. 1.1.99.-) from the ethanol-producing Gram-negative bacterium Zymomonas mobilis is a periplasmic, soluble enzyme that forms a homotetramer of 160 kDa with one NADP(H) cofactor per subunit that is tightly, but noncovalently, bound. The enzyme was crystallized by the hanging drop vapor diffusion method using sodium citrate as precipitant. The obtained crystals belong to the space group P2(1)2(1)2, with unit cell constants of 84.6 A, 94.1 A, and 117.0 A, consistent with two monomers in the asymmetric unit. They diffract to a resolution of about 2 A and are suitable for X-ray structure determination.