Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Pseudomonas fluorescens

Large crystals of arylesterase from Pseudomonas fluorescens have been grown at room temperature using ammonium sulfate as a precipitant. They grow to dimensions of 0.7 × 0.7 × 0.6 mm³ within a month. The crystals belong to the trigonal space group P3₁ (or P3₂), with unit cell dimensions of a= 147.12 Å and c= 131.08 Å. The asymmetric unit seems to contain six molecules of dimeric aryles-terase, with corresponding crystal volume per protein mass (VM ) of 2.53 ų/Da and solvent fraction of 51.5% by volume. The crystals diffract to at least 2.2 Å Bragg spacing when exposed to X-rays from a rotating-anode source. X-ray data have been collected to 2.9 Å Bragg spacing from native crystals. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic analysis of ribosomal protein S8 from Thermus thermophilus

Crystals have been obtained for recombinant ribosomal protein S8 from Thermus thermophilus produced by Escherichia coli. The protein crystals have been grown in 40 mM potassium phosphate buffer (pH 6.0) in hanging drops equilibrated against saturated ammonium sulfate (unbuffered) with 2-methyl-2,4-pentandiol (v/v). The crystals belong to the space group P4₁₍₃₎2₁2 with cell parameters a= b= 67.65 Å, c= 171.12 Å. They diffract x-rays to 2.9 Å resolution. © 1997 Wiley-Liss, Inc.     

Crystallization of the purine salvage enzyme adenine phosphoribosyltransferase

Adenine phosphoribosyltransferase from the protozoan parasite Leishmania donovani has been crystallized in the presence of the substrate Mg²⁺-α-D -5-phosphoribosyl-1-pyrophosphate (PRPP) or the product adenosine-5-monophosphate, as well as in the absence of ligand. These crystals belong to the space group P6₁22 or its enantiomorph P6₅22, with unit cell dimensions of a = b = 64.0 Å, c = 240.5 Å, α = β = 90°, and γ = 120°. The crystals diffract to 1.9 Å. © 1996 Wiley-Liss, Inc.     

Purification,crystallization, and preliminary X-ray studies of 10-formyltetrahydrofolate synthetase from Clostridia acidici-urici

The monofunctional enzyme 10-formyltetrahydrofolate synthetase (THFS), which is responsible for the recruitment of single carbon units from the formate pool into a variety of folate-dependent biosynthetic pathways, has been subcloned, purified, and crystallized. The crystals belong to space group P2₁, with unit cell dimensions a= 102.4 Å b= 116.5 Å c= 115.8 Å and β = 103.5 Å. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme tetramer, and a specific volume of the unit cell of 2.7 Å₃/Da. The crystals diffract to at least 2.3 Å resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. © 1997 Wiley-Liss, Inc.     

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 Å. The crystals belong to the trigonal lattice type with the spacegroup P3₁21 or P3₂21, respectively, have the cell dimensions a = b = 96.8 Å, c = 196.2 Å, α = β = 90°,γ = 120°. The crystals have a theoretical packing density of 2.7 ų/Da, assuming one molecule with a molecular 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. © 1995 Wiley-Liss, Inc.     

Purification,crystallization, and preliminary X-ray studies on the rhizome lectin from stinging nettle and its complex with NN′N″-triacetylchitotriose

Single crystals were grown from affinity-purified stinging nettle lectin and from its complex with the specific trisaccharide NN′N″ -triacetylchitotriose by vapor diffusion at room temperature. The lectin crystallizes in space group P2₁2₁2₁ with unit cell dimensions a = 54.3 (1) Å, b = 62.2 (1) Å, and c = 92.4 (2) Å, and diffracts to 3.0 Å resolution. The asymmetric unit contains three lectin monomers. The crystals of the lectin-trisaccharide complex have space group P2₁2₁2₁ with cell constants a = 37.69 (4) Å, b = 48.97 (6) Å, and c = 57.32 (4) Å. These crystals diffract to at least 2.0 Å resolution and the asymmetric unit contains one lectin monomer. A three-dimensional X-ray structure determination is on its way. © 1993 Wiley-Liss, Inc.     

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.     

Characterization of two crystal forms of Clostridium thermocellum endoglucanase CelC

Endoglucanase CelC from Clostridium thermocellum expressed in Escherichia coli has been crystallized in two different crystal forms by the hanging drop method. Crystals of form I were grown with polyethylene glycol as a precipitant. They are orthorhombic, space group P2₁2₁2₁, with cell dimensions a =51.4 Å, b =84.3 Å, and c =87.5 Å. Crystals of form II, obtained in ammonium sulfate solutions, belong to the tetragonal space group P4₁2₁2 (or P4₃2₁2) with cell dimensions of a = b = 130.7 Å and c = 69.6 Å. Diffraction data to 2.8 Å resolution were observed for both crystal forms with a rotating anode generator. Preliminary oscillation images of the orthorhombic form I crystals using a synchrotron radiation source show diffraction to 2.2 Å resolution, indicating that these crystals are suitable for high resolution crystallographic analysis. © 1994 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray investigation of recombinant human interleukin 10

Crystals of recombinant human interleukin 10 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space group P4₁2₁2 or P4₃2₁2; the unit cell axes are a = 36.5 Å and c = 221.9 Å. There is the equivalent of one polypeptide chain in the asymmetric unit. The crystals are stable to X-rays and diffract to at least 2.5 Å resolution. © 1995 Wiley-Liss, Inc.     

Crystallization and characterization of the recombinant human Clara cell 10-kDa protein

Crystals of recombinant human Clara cell 10-kDa protein were grown both from ammonium sulfate and polyethylene glycol (PEG) solutions. Crystals grown from ammonium sulfate solution have been characterized by X-ray diffraction studies as monoclinic with the space group C2 and lattice constants a = 69.2 Å, b = 83.0 Å, c = 58.3 Å, and β = 99.7°. The monoclinic crystals diffract to beyond 2.5 Å. Some of the crystals grown from PEG were of a similar habit to those grown from ammonium sulfate, but others were triclinic with the space group P1 and cell constants a = 40.3 Å, b = 46.3 Å, c = 51.3 Å, α = 117.7°, β = 102.3°, and γ = 71.4°. These crystals diffract to beyond 3.2 Å. © 1994 Wiley-Liss, Inc.     

Purification,crystallization, and preliminary X-ray diffraction analyses of the bacterial chemotaxis receptor modifying enzymes

Bacterial chemotaxis receptor modifying enzymes from Salmonella typhimurium have been crystallized using microseeding techniques. The crystals of the S-adenosyl-L -methionine-dependent methyl transferase, CheR, belong to the monoclinic space group P2₁ with cell constants a = 55.1 Å, b = 48.1 Å, c = 63.1 Å, β = 112.3°. The crystals of the catalytic domain of the methylesterase, CheB, belong to the trigonal space group P3₂21 or P3₁21 with unit cell dimensions of a = b = 63.4 Å, c = 86.8 Å. Both crystals contain one molecule per asymmetric unit and have calculated Matthews' volumes of 2.4 ų/Da. © 1995 Wiley-Liss, Inc.     

Expression and crystallization of the yeast Hsp82 chaperone,and preliminary x-ray diffraction studies of the amino-terminal domain

Expression of the Saccharomyces cerevisiae Hsp82 chaperone in a pep4-3- and hsc82-deficient strain of S. cerevisiae yielded over 25% of the total cell protein as intact Hsp82. Similarly, the amino-terminal domain (residues 1–220) of Hsp82 was expressed to 18% of the total cell protein. Crystals of the intact Hsp82 were readily obtained. The crystals were very fragile, suggesting a high solvent content, and diffracted to approximately 8 Å. Tetragonal bipyrimidal crystals of the amino-terminal domain of Hsp82 were readily obtained under a variety of different conditions. The crystals have primitive tetragonal space group (P422, P4₁22, or its enantiomorph P4₃22) with unit cell dimensions of a = 75.1 Å and c = 111.3 Å, contain 60% by volume solvent, and diffract to 2.5 Å resolution. Addition of 25% glycerol to the mother liquor gave rise to large rod-shaped crystals. The crystals diffract to 2.8 Å resolution, have an orthorhombic space group (P222₁, P2₁2₁2, or P2₁2₁2₁) with cell dimensions of a = 45.2 Å, b = 115.4 Å, and c = 116.9 Å, and a solvent content of 58% by volume. © 1996 Wiley-Liss, Inc.     

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 of Thermus thermophilus histidyl-tRNA synthetase and Its complex with tRNAHis

Histidyl-tRNA synthetase (HisRS) has been purified from the extreme thermophile Thermus thermophilus. The protein has been crystallized separately with histidine and with its cognate tRNA^His. Both crystals have been obtained using the vapor diffusion method with ammonium sulphate as precipitant. The crystals of HisRS with histidine belong to the spacegroup P2₁2₁2 with cell parameters a = 171.3 Å, b = 214.7 Å, c = 49.3 Å, α = β = γ = 90°. A complete data set to a resolution of 2.7Å with an R_merge on intensities of 4.1% has been collected on a single frozen crystal. A partial data set collected on a crystal of HisRS in complex with tRNA_His shows that the crystals are tetragonal with cell parameters a = b = 232 Å, c = 559 Å, α = β = γ = 90° and diffract to about 4.5 Å resolution. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray analysis of the cytoplasmic domain of human erythrocyte band 3

A cytoplasmic domain of the human erythrocyte membrane protein band 3 (M_r = 42,500), residues 1–379, expressed in and purified from E. coli, has been crystallized by the method of vapor diffusion in sitting drops with subsequent streak-seeding at room temperature. Initial crystals were grown from solutions containing 65–68% saturated ammonium sulfate at pH 4.9 and 2 mg/ml protein. Subsequent streak-seeding into solutions of 50–53% ammonium sulfate at pH 4.9 and 7 mg/ml protein produced single crystals suitable fur X-ray analysis, which contained pure protein as revealed by gel electrophoresis. The crystals belong to the monoclinic space group C2 with cell dimensions of a = 178.8 Å, b = 90.5 Å, c = 122.1 Å, and β = 131.3° and diffract at least to 2.7 Å resolution (at 100 K). A self-rotation function shows the presence of approximate 222 local symmetry. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary diffraction analysis of Ca2+-calmodulin-drug and apocalmodulin-drug complexes

Ca²⁺-calmodulin is crystallized with two new and potent drugs: a bisindol derivative (KAR-2, 3”-(β-chloroethyl)-2”,4”-dioxo-3,5”-spiro-oxazolidino-4-deacetoxy-vinblastine) with antitumor activity and an arylalkylamine fendiline analogue (N-(3,3-diphenylpropyl)-N'-[1-(3,4-di-n-butoxy-phenyl)-ethyl]-1,3-diaminopropane) with anticalmodulin activity. The crystals diffract beyond 2.8 Å and differ in unit cell parameters from each other as well as from crystals of Ca²⁺-calmodulin or Ca²⁺-calmodulin-ligand complexes, as reported thus far. Attempts to crystallize Ca²⁺-free calmodulin without drugs failed, in consonance with earlier results; however, single Ca²⁺-free calmodulin crystals diffracting beyond 2.5 Å resolution were grown in the presence of KAR-2. Results indicate that binding of the two drugs to apocalmodulin or Ca²⁺-calmodulin may induce unique novel protein conformers, targets of further detailed X-ray studies. © 1997 Wiley-Liss Inc.     

Crystallization and preliminary crystallographic data of a neutrophil migration-inducing lectin (KM+) extracted from the seed of Artocarpus integrifolia

The tetrameric KM+ lectin from the seeds of Artocarpus integrifolia has, when compared to other plant lectins, the singular property of directly inducing neutrophil migration into the peritoneal cavity or into the air pouch of rats. This protein crystals have been grown and they belong to the orthorhombic system with space group C222₁. The unit cell parameters are a = 54.4 Å, b = 127.9 Å and c = 99.8 Å. A native diffraction dataset to 2.8 Å was collected and an analysis of the self-rotation function has shown the presence of only one independent non-crystallographic 2-fold axis orthogonal to the crystal b-axis, compatible with a dimer in the asymmetric unit. Proteins 27:157–159 © 1997 Wiley-Liss, Inc.     

Purification,crystallization, and preliminary X-ray analysis of PepX,an X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis

The X-prolyl dipeptidyl aminopeptidase PepX, a serine peptidase isolated originally from Lactococcus lactis subsp lactis NCDO 763, was cloned and overproduced in Escherichia coli. The enzyme was isolated in its active form in two purification steps. Crystals of PepX were grown by the hanging drop vapor diffusion method using polyethyleneglycol 4000 as precipitant at pH 5.0. The crystals are orthorhombic with cell dimensions a = 92.8 Å, b = 102.6 Å, and c = 101.6 Å, space group P2₁2₁2, and probably contain one monomer of 87.5 kDa in the asymmetric unit. The crystals, very stable under X-rays, diffract to at least 2.2 Å and are suitable for high-resolution structural analysis. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray analysis of nonglycosylated tissue inhibitor of metalloproteinases-1, N30QN78Q TIMP-1

A nonglycosylated (N₃₀QN₇₈Q) form of the human tissue inhibitor of metalloproteinases, TIMP-1, has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small single crystals have been grown using sodium tartrate as a precipitant. The crystals are in space group P2₁, with cell dimensions a = 35.28, b = 53.95, c = 48.56, and β = 96.0°. There is a single molecule of TIMP-1 in the asymmetric unit. The crystals diffract to at least 2.3 Å resolution. Complete data have been collected to 2.9 Å and a search for heavymetal derivatives is in progress. © 1993 Wiley-Liss, Inc.