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.     

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.     

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 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,characterization, and preliminary crystallographic studies of mitochondrial carbamoyl phosphate synthetase I of Rana catesbeiana

Carbamoyl phosphate synthetase I (ammonia; E C 6.3.4.16) was purified from the liver of Rana catesbeiana (bullfrog). Crystals of the protein have been obtained at 22°C by the hanging drop vapor diffusion technique, with polyethylene glycol as precipitant. Tetragonal crystals of about 0.3 × 0.3 × 0.7 mm diffract at room temperature to at least 3.5 Å using a conventional source and are stable to X-radiation for about 12 h. Therefore, these crystals are suitablefor high resolution studies. The space group is P4₁2₁2 (or its enantiomorph P4₃2₁2), with unit cell dimensions a = b = 291.6 Å and c = 189.4 Å. Density packing considerations areconsistent with the presence of 4-6 monomers (M_r of the monomer, 160,000) in the asymmetric unit. Amino-terminal sequence of the enzyme and of a chymotryptic fragment of 73.7 kDa containing the COOH-terminus has been obtained. The extensive sequence identity with rat and human carbamoyl phosphate synthetase I indicates the relevance for mammals of structural data obtained with the frog enzyme. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic data for class I deoxyribose-5-phosphate aldolase from Escherichia coli: An Application of Reverse Screening

X-ray quality crystals of class I deoxyribose-5-phosphate aldolase from Escherichia coli have been obtained for the unliganded enzyme and in complex with its substrate, 2-deoxyribose-5-phosphate. The enzyme catalyzes the reversible cleavage of 2-deoxyribose-5-phosphate to acetaldehyde and D-glyceraldehyde-3-phosphate. The unliganded and complex crystals are prismatic long rods and belong to the orthorhombic space group P2₁2₁2₁ with cell dimensions a = 183.1 Å, b = 61.4 Å, c = 49.3 Å and a = 179.2 Å, b = 60.5, Å, c = 49.1 Å, respectively. Two molecules in the asymmetric unit are related by a noncrystallo-graphic 2-fold axis. The crystals are stable in the X-ray beam and diffract to at least 2.6 Å. A new method, reverse screening, designed to minimize protein utilization during the screening process was used to determine supersaturation and crystallization conditions. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray diffraction studies of an a1/α2/DNA ternary complex

Crystals have been obtained of a ternary complex containing the yeast a1/α2 homeodomain heterodimer bound to a 21-base pair DNA site containing two 5′ overhanging bases at each end. The crystals are grown from cobaltic hexamine and form in space group P6₁ or P6₅ with a = b = 133 Å, c = 45.4 Å. Crystals that are flash-frozen at ?179°C diffract to 2.7 Å along the c-axis and to 2.4 Å in perpendicular directions. The crystals contain one protein–DNA complex in the crystallographic asymmetric unit. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of ImmE7 protein of colicin E7

The ImmE7 protein, which can bind specifically to the DNase colicin E7 and neutralize its bactericidal activity, has been purified and crystallized in two different crystal forms by vapor diffusion method. The orthorhombic crystals belong to space group I222 or I2₁2₁2₁ and have unit cell dimensions a = 75.1 Å, b = 50.5 Å, and c = 45.4 Å. The second form is monoclinic space group P2₁ with ceil dimensions a = 29.3 Å, b = 102.7 Å, c = 53.0 Å and β = 91.5°. The orthorhombic crystals diffract to 1.8 Å resolution, and are suitable for high-resolution X-ray analysis. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic studies of the precursor and mature forms of a neutral lipase from the fungus Rhizopus delemar

A neutral lipase from the filamentous fungus Rhizopus delemar has been crystallized in both its proenzyme and mature forms. Although the latter crystallizes readily and produces a variety of crystal forms, only one was found to be suitable for X-ray studies. It is monoclinic (C2, a = 92.8 Å, b = 128.9 Å, c = 78.3 Å, β = 135.8) with two molecules in the asymmetric unit related by a noncrystallographic diad. The prolipase crystals are orthorhombic (P2₁2₁2₁, with a = 79.8 Å, b = 115.2 Å, c = 73.0 Å) and also contain a pair of molecules in the asymmetric unit. Initial results of molecular replacement calculations using the refined coordinates of the related lipase from Rhizomucor miehei identified the correct orientations and positions of the protein molecules in the unit cells of crystals of both proenzyme and the mature form. © 1994 John Wiley & Sons, 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.     

Preparation and initial characterization of crystals of the photoprotein aequorin from Aequorea victoria

Crystals of recombinant aequorin, the photoprotein from the jellyfish Aequorea victoria, have been grown from solutions containing sodium phosphate. The crystals grow as thin plates which diffract to beyond 2.2 Å resolution. The crystals are orthorhombic, space group P2₁2₁2 ₁; the axes are a = 89.1(1), b = 88.4(1), and c = 52.7(1) Å. The asymmetric unit contains two molecules. Crystals exposed to calcium ion solutions emit a steady glow and slowly deteriorate, confirming that the crystals consist of a charged, competent photoprotein. This represents the first successful preparation of single crystals of a photoprotein suitable for diffraction analysis. © 1993 Wiley-Liss, Inc.     

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.     

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 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.     

Crystallization and preliminary X-ray crystallographic studies of ketosteroid isomerase from Pseudomonas putida biotype B

The Δ⁵-3-ketosteroid isomerase from Pseudomonas putida biotype B has been crystallized. The crystals belong to the space group P2₁2₁2₁ with unit cell dimensions of a = 36.48 Å, b = 74.30 Å, c = 96.02 Å, and contain one homodimer per asymmetric unit. Native diffraction data to 2.19 Å resolution have been obtained from one crystal at room temperature indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.     

Crystallization of the reaction center from Rhodobacter sphaeroides in a new tetragonal form

The reaction center from the nonsulfur purple bacteriumRhodobacter sphaeroideshas been crystallized in a new form. The crystals grew in the presence of polyethylene glycol 4000, the detergent β-octyl glucoside, and the amphiphiles heptane triol and benzamidine hydrochloride, using the sitting drop method. The space group of these crystals is tetragonal, P4₁(4₃)2₁2, and the cell constants are a = b = 141.5 Å and c = 276.7 Å with probably 2 proteins per asymmetric unit. A native data set has been set collected to a resolution of 2.8 Å consisting of 56,332 unique reflections (50,731 with F > 2σ) with anR_sym of 9.5%. Analysis of the diffraction data is underway using molecular and isomorphous replacement. © 1994 Wiley-Liss, Inc.     

Crystallization and X-ray analysis of a single fab binding domain from protein L of Peptostreptococcus magnus

Protein L is a multi domain cell wall constituent of certain strains of Peptostreptococcus magnus which binds to the variable domain of immunoglobulin κ-light chains. A single immunoglobulin-binding domain of M_r = 9000 from this protein has been isolated and crystallized. The crystals are of space group P4₂2₁2, with cell dimensions a = b = 66.9 Å, c = 68.3 Å, and diffract to at least 2.2 Å resolution. The asymmetric unit of the crystal contains two molecules of the protein L domain, related by a noncrystallographic 2-fold axis, as revealed by a self-rotation function calculated with native diffraction data. © 1995 Wiley-Liss, Inc.     

Crystallization of the bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase domain of the human trifunctional enzyme

Methylenetetrahydrofolate([H₄] folate) dehydrogenase (D) and methenyl[H₄] folate cyclohydrolase (C) coexist as a bifunctional enzyme (DC) or as the amino-terminal domain of a trifunctional enzyme (DCS) where the third activity is 10-formyl[H₄]lfolate synthetase (S). Two crystal forms of the DC domain of the human cytosolic DCS enzyme have been grown from polyethyleneglycol solution. The monoclinic P2₁ crystals diffract to 2.8 Å with a = 72.5 Å, b = 68.5 Å, c = 125.2 Å, and β = 91.8° but were found to be twinned. The orthorhombic P2₁2₁2₁ crystals diffract to 2.5 Å with a = 67.7 Å, b = 135.9 Å, c = 61.6 Å, and contain two molecules per asymmetric unit. Proteins 26:479–480 © 1996 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.     

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.