Crystallization and preliminary X-ray diffraction study of an endoglucanase from Clostridium thermocellum

Endoglucanase D, a cellulose degradation enzyme from Clostridium thermocellum has been cloned in Escherichia coli, purified and crystallized. The crystals are trigonal, space group P3(1)12 (or P3(2)12) with a = 57.7 (+/- 0.1) A, c = 192.1 (+/- 0.2) A, and diffract X-rays to a resolution of 2.8 A. They are suitable for a high-resolution X-ray diffraction analysis.     

Crystallization and preliminary x-ray analysis of the flavoenzyme vanillyl-alcohol oxidase from Penicillium Simplicissimum

Vanillyl-alcohol oxidase catalyses the oxidation of several 4-hydroxybenzyl alcohols by using 8-α-(N³-histidyl)-FAD as a covalently bound prosthetic group. Crystals of vanillyl-alcohol oxidase from Penicillium simplicissimum have been grown by using the vapor diffusion technique. The space group was found to be I4, with cell dimensions a = b = 140.5 Å, c = 132.9 Å. Diffraction data have been recorded to 3.2 Å resolution by using a laboratory source and to 2.5 Å resolution on flash freezing the crystal at the ELETTRA Synchrotron X-ray diffraction beam line. Proteins 27:601–603, 1997. © 1997 Wiley-Liss Inc.     

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 studies of human salivary alpha-amylase.

Nonglycosylated alpha-amylase, a major component of human parotid saliva, has been crystallized by the vapor diffusion technique using 2-methyl-2,4-pentanediol as the precipitant in the presence of CaCl2 at pH 9.0. The crystals are orthorhombic, space group P2(1)2(1)2(1) with unit cell dimensions of a = 53.3, b = 75.8, and c = 138.1 A. The asymmetric unit contains one amylase molecule. The solvent content is 54%. The crystals are stable to X-rays and diffract up to 2.8 A and appear to be suitable for X-ray diffraction studies.     

Crystallization and preliminary X-ray analysis of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATP gamma S, fructose 6-phosphate, the detergent n-octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 A and c = 130.6 A. Flash-frozen crystals diffract to beyond 2.2 A, and native data have been collected.     

Crystallization and preliminary X-ray diffraction study of a xylanase from Trichoderma harzianum

A 20,000 Mr xylanase from Trichoderma harzianum has been purified and crystallized from 20% (w/v) saturated ammonium sulphate solutions. The unit cell is orthorhombic, space group P2(1)2(1)2(1), with unit cell lengths a = 44.2 A, b = 94.1 A, c = 51.6 A. Data from native crystals and several potential heavy-atom derivatives have been collected. An X-ray analysis to at least 2.8 A resolution appears to be feasible.     

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 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.     

Structure of presynaptic toxins: Crystallization and preliminary x-ray diffraction data on Notechis II-5, a presynaptic toxin phospholipase

The presynaptic neutrotoxin-phospholipase, Notechis II-5 from the venom of NotechisScutatus scutatus (Australian tiger snake) has been crystallized in a form suited for x-ray diffraction analysis. The crystals belong to the orthorhombic space group P2₁ 2₁,2₁, with unit cell dimensions, a=146.1,b =43.5 and c =39.0 A. There are two molecules of Notechis II-5 in the asymmetric unit. The molecular weight is about 13,500. Notechis II-5 is highly homologous to Notexin, another presynaptic toxin from the venom of the Australian tiger snake, to bovine and porcine pancreatic phospholipases A and other venom phospholipases.     

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 analysis of 5,10- methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Thermoplasma acidophilum DSM 1728

The methylenetetrahydrofolate dehydrogenase/ cyclohydrolase (MTHFDC) from the thermoacidophilic archaeon Thermoplasma acidophilum is a 30.6 kDa molecular-mass enzyme that sequentially catalyzes the conversion of formyltetrahydrofolate to methylenetetrahydrofolate, with a preference for NADP as a cofactor, rather than NAD. In order to elucidate the functional and structural features of MTHFDC from archaeons at a molecular level, it was overexpressed in Escherichia coli and crystallized in the presence of its cofactor, NADP, at 295 K using polyethylene glycol (PEG) 4000 as a precipitant. The crystal is a member of the monoclinic space group P21, with the following unit cell parameters: a=66.333 A, b=52.868 A, c=86.099 A, and beta= 97.570o, and diffracts to a resolution of at least 2.40 A at the synchrotron. Assuming a dimer in the crystallographic asymmetric unit, the calculated Matthews parameter (VM) was 2.44 A3/Da and the solvent content was 49.7%.     

Crystallization and preliminary X-ray diffraction study of 1,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea

1,3,8-Trihydroxynaphthalene reductase was crystallized in the presence of NADPH and the inhibitor tricyclazole. The crystals are trigonal, space group P3₁21 or its enantiomorph P3₂21. Two crystal forms with slightly different cell dimensions were obtained. Form A has unit cell dimensions a = b = 142.6 Å, c = 70.1 Å and form B cell dimensions a = b = 142.6 Å, c = 72.9 Å. The diffraction pattern of the latter crystal form extends to 2.5 Å resolution.     

Crystallization and preliminary X-ray diffraction study of ferrocytochrome c2 from Rhodopseudomonas viridis

Crystals of ferrocytochrome c2 from a non-sulphur purple photosynthetic bacterium, Rhodopseudomonas viridis, have been grown from ammonium sulphate solution at pH 8.5 by the sitting-drop vapour-diffusion procedure. The crystals belong to the trigonal system, space group P3(1)21 (or its enantiomorph P3(2)21) with unit-cell dimensions of a = b = 75.8 A and c = 40.1 A, and diffract to at least 2.0 A resolution. Assuming that an asymmetric unit contains one protein molecule (approx. 12,300 Mr), the solvent content of the crystal is approximately 54.5% (v/v).     

Expression,crystallization and preliminary X-ray diffraction study of FtsY,the docking protein of the signal recognition particle of E. coli

FtsY is the docking protein or SRα homologue in E. coli. It is involved in targeting secretory proteins to the cytoplasmic membrane by interacting with the signal recognition particle, controlled by guanosine 5′-triphosphate. Two different constructs have been used in crystallization studies: the full-length protein and a truncated fragment with a his-tag at the C terminus. Only the second construct resulted in crystals suitable for x-ray diffraction. The crystals belong to the monoclinic space group P2₁ with cell dimensions a = 32.20 Å, b = 79.57 Å, c = 59.21 Å, and β = 94.45, and contain one molecule per asymmetric unit. At cryogenic temperatures the crystals diffract to a resolution limit of 2.5 Å by using a rotating anode, and beyond 1.8 Å by using synchrotron radiation. Proteins 28:285–288, 1997. © 1997 Wiley-Liss Inc.     

Crystallization and preliminary X-ray diffraction studies of the lectin from Erythrina corallodendron

The lectin from Erythrina corallodendron, specific for N-acetyllactosamine, crystallizes in the hexagonal space group P6(1) (P6(5)) with unit cell dimensions a = b = 136.3 A, c = 83.2 A and one dimer of Mr 60,000 in the asymmetric unit. The crystals are suitable for high-resolution work.     

Crystallization and preliminary X-ray diffraction data of two different human low-density lipoprotein (LDL) subfractions

Human LDL subfractions LDL-2 (d = 1.031–1.034 g/ml) and LDL-5 (d = 1.040–1.044 g/ml) were crystallized in two different crystal forms by using polyethylene glycol as a precipitant. Both fractions were from one donor. Crystals of LDL-5 were yellow, hexagonal, and showed no dichroism. Crystals of LDL-2 were of the same color, had a rodlike shape with notches at both ends, and were highly dichroitic. LDL-2 crystals diffracted to a resolution of 29 Å by using synchrotron radiation. Indexing in P1 resulted in preliminary parameters for the reduced cell of a = 171 Å, b = 438 Å, c = 519 Å, α = 102°, β = 99°, γ = 91. These dimensions are consistent with the size of LDL particles. Using Fourier transform infrared spectroscopy (FTIR) and agarose gel electrophoresis, we could further confirm that the crystals consist of LDL. The FTIR spectrum showed bands characteristic for lipids and protein. Dissolved crystals exhibited a mobility similar to native LDL in agarose gels and could be stained with anti-human apolipoprotein B (apoB). Proteins 28:293–297, 1997. © 1997 Wiley-Liss Inc.     

Crystallization and preliminary X-ray diffraction study of a protein with a high potential rubredoxin center and a hemerythrin-type Fe center

A newly discovered iron-containing protein, isolated from the bacterium Desulfovibrio vulgaris (Hildenborough, NCIB 8303), has been crystallized. The molecule appears to be a dimer of mass 44kDa. This protein has iron centers with spectrascopic similarities to those in rubredoxins and in hemerythrins. The X-ray diffraction shows symmetry consistent with space group I222 or I212121. Cell parameters are a = 49.2 A, b = 81.3 A, c = 100.1 A, and alpha, beta, gamma = 90 degrees. X-ray diffraction data have been collected to 3.0 A, and a search for useful heavy atom derivatives is in progress for the analysis of the crystal structure of this Fe-protein.     

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.     

Crystallization and preliminary X-ray crystallographic analysis of chitinase from barley seeds

Chitinase from barley seeds has been crystallized at room temperature using polyethylene glycol as precipitant. The crystal is monoclinic, belonging to the space group P2₁, with unit cell parameters of a = 69.43 Å, b = 44.55 Å, c = 81.41 Å, and β = 111.95 Å. The asymmetric unit seems to contain two molecules of chitinase with a corresponding crystal volume per protein mass (V_M) of 2.25 ų/Da and a solvent content of 45% by volume. The crystal diffracts to at least 2.0 Å with X-rays from a rotating anode source and is very stable in the X-ray beam. X-ray data have been collected to better than 2.2 Å Bragg spacing from a native crystal. © 1993 Wiley-Liss, Inc.     

Crystallization of 5-aminolaevulinic acid dehydratase from Escherichia coli and Saccharomyces cerevisiae and preliminary X-ray characterization of the crystals.

5-Aminolaevulinic acid dehydratase (ALAD) catalyzes the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. Both Escherichia coli and Saccharomyces cerevisiae ALADs are homo-octameric enzymes which depend on Zn2+ for catalytic activity and are potently inhibited by lead ions. The E. coli enzyme crystallized in space group I422 (unit cell dimensions a = b = 130.7 A, c = 142.4 A). The best crystals were obtained in the presence of the covalently bound inhibitor laevulinic acid. The yeast enzyme (expressed in E. coli) crystallized in the same space group (I422) but with a smaller unit cell volume (a = b = 103.7 A, c = 167.7 A). High resolution synchrotron data sets were obtained from both E. coli and yeast ALAD crystals by cryocooling to 100 K.