Crystals of the complex between recombinant N-acetyleglin c and subtilisin. A preliminary characterization

The complex between recombinant N-acetyleglin c and subtilisin has been crystallized into a triclinic cell. The cell constants are a = 38.3 A, b = 41.5 A, c = 56.6 A, alpha = 110.6 degrees, beta = 83.8 degrees, and gamma = 104.7 degrees. The crystals diffract to better than 2.3-A resolution and are large enough for data collection. The crystallization conditions and general crystal data are presented.     

Crystallization and preliminary diffraction analysis of cholesterol esterase from Candida cylindracea

Cholesterol esterase (EC 3.1.1.13) from the microorganism Candida cylindracea has been crystallized in two forms. Crystals, typically 0.30 x 0.15 x 0.10 mm in size, diffract rotating anode generated x-rays to beyond 3 A are suitable for data collection for an x-ray crystallographic investigation. A monoclinic crystal form in the space group P2(1) was found to have cell dimensions of a = 122.9 A, b = 101.0 A, c = 95.2 A and beta = 108.3 degrees. The asymmetric unit of the cell contains two dimers of 129 kDa each. A second crystal form, in the triclinic space group P1, has cell dimensions of a = 58.6 A, b = 88.7 A, c = 58.6 A, alpha = 93.3 degrees, beta = 113.8 degrees and gamma = 96.0 degrees, and has one dimer per asymmetric unit.     

Crystal structures of recombinant and native soybean beta-conglycinin beta homotrimers.

The crystal structures of recombinant and native beta homotrimers of soybean beta-conglycinin were determined by X-ray crystallography at 2.7 and 2.8 A resolutions, respectively. The crystals of the recombinant and native beta homotrimers belong to space group P21 with cell parameters a = 80.51 A, b = 63.48 A, c = 131.43 A, and beta = 90.01 degrees and with cell parameters a = 82.78 A, b = 69.47 A, c = 125.33 A and beta = 97.22 degrees, respectively. The beta monomers consist of amino-terminal and carboxyl-terminal modules that are very similar to each other and are related by a pseudo-dyad axis. Each module of the beta monomer is subdivided into a core and a loop domain. These structural features of both beta homotrimers are consistent with those of canavalin and phaseolin, which are similar vicilin class proteins. The superposition of the models of the native and recombinant beta monomers shows a root mean square deviation of 0.43-0.51 A for 343 common Calpha atoms within 2.0 A. This result indicates that the N-linked glycans do not influence the final structure of the beta homotrimer. Comparison of the models of beta-conglycinin, phaseolin and canavalin indicates that beta-conglycinin resembles canavalin rather than phaseolin, and that canavalin and phaseolin differ the most among them. The evolutional relationships are discussed.     

Crystallization and preliminary X-ray analysis of flavocytochrome c(3), the fumarate reductase from Shewanella frigidimarina.

The fumarate reductase (flavocytochrome c(3)) from Shewanella frigidimarina (formerly S. putrefaciens) NCIMB400 has been crystallized in the space group P2(1), with cell dimensions of a = 45.447 A, b = 92.107 A, c = 78.311 A, and beta = 91.038 degrees and one molecule per asymmetric unit. A native data set has been collected to 1.8 A. The gene encoding Fcc(3) from the S. frigidimarina type strain ACAM591 has been cloned and sequenced and the protein crystallized in space group P2(1) with cell dimensions of a = 45.359 A, b = 88.051 A, c = 77.473 A, and beta = 104.499 degrees. Anomalous data have also been collected from the NCIMB400 crystal allowing the heme iron positions to be identified.     

The lumazine synthase-riboflavin synthase complex of Bacillus subtilis. Crystallization of reconstituted icosahedral beta-subunit capsids

The lumazine synthase-riboflavin synthase complex (heavy riboflavin synthase) of Bacillus subtilis consists of an icosahedral capsid of 60 beta-subunits containing a core of three alpha-subunits. The enzyme has been purified from the derepressed mutant H 94 of B. subtilis by a novel efficient procedure using column chromatography and preparative crystallization. Beta-Subunits were isolated after dissociation of the enzyme at pH 8.0. Ligand-driven renaturation of beta-subunits yields hollow icosahedral beta 60 capsids which could be crystallized in 1.55 M phosphate, pH 8.7, in three different modifications. A monoclinic modification belongs to space group C2 with unit cell dimensions of a = 235.5, b = 191.2, and c = 165.4 A and alpha = gamma = 90 degrees and beta = 134.4 degrees. The crystals contain two hollow beta 60 particles/unit cell and diffract to approximately 2.8-A resolution. A hexagonal modification has the space group P6(3)22 with unit cell dimensions of a = b = 157.2 and c = 300.8 A and alpha = beta = 90 degrees and gamma = 120 degrees. These cell parameters are similar to the dimensions of hexagonal crystals of native heavy riboflavin synthase (alpha 3 beta 60). A second hexagonal modification shows unit cell parameters of a = b = 156.3 and c = 622.6 A and alpha = beta = 90 degrees and gamma = 120 degrees. The space group of this modification could not be determined unambiguously.     

Crystallization of a ternary complex of lactate dehydrogenase from Bacillus stearothermophilus

Bacillus stearothermophilus lactate dehydrogenase was purified from an overexpressing Escherichia coli cell line. The enzyme has been crystallized in several different forms. All of these crystal forms were grown in the presence of NADH, sodium oxamate and fructose 1,6-bisphosphate. Three crystal forms have been characterized, an orthorhombic P2(1)2(1)2 (type III, a = 86 A, b = 105 A, c = 136 A) and two monoclinic P21 forms (type IV, a = 85 A, b = 118 A, c = 136 A, beta = 96 degrees; type V, a = 112 A, b = 85 A, c = 136 A, beta = 91 degrees). Precession photographs from these crystal forms are very alike, suggesting the molecular packing to be similar in all three forms. The P21 type IV crystals diffract to beyond 2 A spacing and are stable to irradiation with X-rays. A complete medium-resolution (4.7 A) dataset has been collected from a single crystal using synchrotron radiation. Rotation function studies with these data show the two tetramers of the asymmetric unit to be in very similar orientations. Higher-resolution data are being collected.     

Crystallization and preliminary crystallographic studies of the recombinant antitumour lectin from the edible mushroom Agrocybe aegerita

The antitumour lectin from Agrocybe aegerita, named AAL, shows strong inhibition effects on human and mouse tumour cells via apoptosis induction activity. Recombinant AAL (rAAL) has been expressed and purified. Both rAAL and rAAL-lactose complex have been crystallized and their X-ray diffraction data were collected to resolutions of 1.9 A and 1.6 A, respectively. Both crystals belong to space group P2(1) with unit cell parameters a = 53.20 A, b = 66.01 A, c = 57.86 A, beta = 109.38 and a = 53.38 A, b = 66.29 A, c = 58.02 A, beta = 109.03, respectively.     

Crystallization of human beta-hexosaminidase B.

beta-Hexosaminidase is a lysosomal hydrolase that is important in the metabolism of sphingoglycolipids. beta-Hexosaminidase B and beta-hexosaminidase A are the major isozymes in normal human tissue. beta-Hexosaminidase B is a homodimer of beta subunits, and beta-hexosaminidase A is a heterodimer composed of an alpha and a beta subunit. Crystals of beta-hexosaminidase B (M(r) 112,000) have been grown using the handling drop technique. They are elongated hexagonal prisms with maximum dimensions of 0.2 mm x 0.2 mm x 0.7 mm. The space group is P6(1)22 (or enantiomorph); the unit cell dimensions are a = b = 114.2 A, c = 402.2 A, alpha = beta = 90 degrees, gamma = 120 degrees. The molecular mass and cell dimensions suggest that there is one dimer per asymmetric unit. Crystals diffract to 3.2 A resolution.     

Preliminary crystallographic analysis of trypanothione reductase from Crithidia fasciculata

Trypanothione reductase, a flavoprotein disulfide reductase specific to trypanosomatid parasites, has been crystallized by vapor diffusion of a protein solution (10 mg/ml) against 22% polyethylene glycol (average Mr 8000) containing 100 mM-ammonium sulfate. Crystals of a size suitable for structure determination by X-ray diffraction have been obtained by seeding protein solutions with smaller crystals. The space-group is P21 (a = 60.9 A, b = 161.8 A, c = 58.4 A, beta = 99.1 degrees). The molecular mass and volume of the unit cell suggest that there is a dimer of the enzyme in the asymmetric unit, and this is confirmed by self-rotation functions calculated using data to 4.5 A resolution. The crystals diffract to beyond 3 A resolution. Crystals of another P21 form (a = 91.3 A, b = 114.4 A, c = 92.0 A, beta = 141.3 degrees) are observed to grow under similar conditions.     

Two crystal forms of the lentil lectin diffract to high resolution.

The legume lectins are an important class of polysaccharide-binding proteins with a wide range of biochemical and immunological applications. Two high-resolution crystal forms are obtained for the lentil (Lens culinaris) lectin: a monoclinic P21 and an orthorhombic P212121. The unit cell dimensions for the monoclinic form are a = 58.0 A, b = 56.0 A, c = 82.1 A, beta = 104.4 degrees, while for the orthorhombic form a = 56.4 A, b = 74.6 A, c = 124.9 A. The asymmetric unit contains one dimer in both cases. The crystals diffract to 1.7 A resolution using synchrotron radiation. Preliminary data have been collected to 2.3 A on both crystal forms using a conventional X-ray source.     

Crystallographic study of endogenous alpha-amylase inhibitor from wheat

Endogenous alpha-amylase inhibitor from wheat has been crystallized by a microdialysis method. There are two forms of monoclinic crystal in a microdialysis cell with a space group of P2(1). The unit cell dimensions are a = 43.5 A, b = 64.8 A, c = 32.2 A, beta = 113 degrees for the rod-like crystal, and a = 42.5 A, b = 65.2 A, c = 32.2 A, beta = 112 degrees for the plate-like crystal. The former is suitable for structure analysis because it gives the sharp diffraction beyond 2.0 A resolution, and the latter tends to form a twin crystal. A heavy-atom derivative has been successfully prepared with the heavy-atom reagent K2PtCl4, and structure analysis is in progress.     

Crystallization and preliminary X-ray diffraction studies of a toxic phospholipase A2 from the venom of Vipera ammodytes meridionalis complexed to a synthetic inhibitor

A toxic phospholipase A(2) (PLA(2)) is isolated from the neurotoxic complex Vipoxin, the major lethal component of the venom of Vipera ammodytes meridionalis. The enzyme is complexed to the synthetic inhibitor elaidoylamide and crystallized. The crystals belong to the space group P2(1)2(1)2(1), with unit cell dimensions a=46.57 A, b=82.68 A, c=119.47 A and beta=90 degrees. Initial diffraction data to 3.3 A resolution are collected.     

Crystallization and preliminary X-ray crystallographic analysis of Ace: a collagen-binding MSCRAMM from Enterococcus faecalis

Ace is a collagen-binding bacterial cell surface adhesin from Enterococcus faecalis. The collagen-binding domain of Ace (termed Ace40) and its truncated form Ace19 have been crystallized by the vapor-diffusion hanging-drop method. Ace19 was crystallized in two different crystal forms. A complete 1.65 A data set has been collected on the orthorhombic crystal form with unit cell parameters a=38.43 b=48.91 and c=83.73 A. Ace40 was crystallized in the trigonal space group P3(1)21 or P3(2)21 with unit cell parameters a=b=80.24, c=105.91 A; alpha=beta=90 and gamma=120 degrees. A full set of X-ray diffraction data was collected to 2.5 A. Three heavy atom derivative data sets have been successfully obtained for Ace19 crystals and structural analysis is in progress.     

Preliminary X-ray crystallographic study of cyanase from Escherichia coli

Cyanase, an oligomeric enzyme of Escherichia coli that catalyzes the decomposition of cyanate to ammonia and bicarbonate, crystallizes in the space group P1 with unit cell parameters a = 85.96 A, b = 83.17 A, c = 83.28 A, alpha = 110.29 degrees, beta = 118.29 degrees and gamma = 72.40 degrees. Crystals diffract to a resolution of at least 2.5 A. The crystal data, in conjunction with a subunit molecular weight of 17,008, suggest that two oligomers are in the asymmetric unit of the crystal and that eight subunits comprise a single oligomer.     

Isolation, crystallization, crystal structure analysis and refinement of B-phycoerythrin from the red alga Porphyridium sordidum at 2.2 A resolution.

The light-harvesting pigment-protein complex B-phycoerythrin from the red alga Porphyridium sordidum has been isolated and crystallized. B-Phycoerythrin consists of three different subunits forming an (alpha beta)6 gamma aggregate. The three-dimensional structure of the (alpha beta)6 hexamer was solved by Patterson search techniques using the molecular model of C-phycocyanin from Fremyella diplosiphon. The asymmetric unit of the crystal cell (space group P3, with a = b = 111.2 A, c = 59.9 A, alpha = beta = 90 degrees, gamma = 120 degrees) contains two (alpha beta) monomers related by a local dyad. Three asymmetric units are arranged around the crystallographic 3-fold axis building an (alpha beta)6 hexamer, as in C-phycocyanin. The crystal structure has been refined by energy-restrained crystallographic refinement and model building. The conventional R-factor of the final model was 18.9% with data to 2.2 A resolution. The molecular structures of the alpha and beta-subunits resemble those of C-phycocyanin. Major changes in comparison to phycocyanin are caused by deletion or insertion of segments involved in protein-chromophore interactions. The singly linked phycoerythrobilin chromophores alpha-84, alpha-140a, beta-84 and beta-155 are each covalently bound to a cysteine by ring A. The doubly linked chromophore beta-50/beta-61 is attached at cysteine beta-50 through ring A and at cysteine beta-61 through ring D. B-Phycoerythrin contains additionally a 30 kDa gamma-subunit, which is presumably located in the central cavity of the hexamer. It is disordered, as a consequence of crystal and local symmetry averaging.     

Preliminary X-ray crystallography studies of recombinant human interleukin-1 alpha. Purification and structural characterization

Human interleukin-1 alpha, cloned and expressed in E. coli, has been purified and structurally characterized by various physiochemical methods, including mass spectrometry. The recombinant protein has been crystallized by the hanging drop vapor diffusion method using dimethyl sulfoxide as the precipitating agent. The space group is P2(1)2(1)2(1). Unit cell dimensions are a = 44.1, b = 57.1, and c = 61.7 A and alpha = beta = gamma = 90 degrees. The crystals diffract to beyond 1.7 A and are suitable for high resolution data collection. Native diffraction data were collected. Screens for heavy atom derivatives have been initiated.     

X-ray studies on triclinic crystals of fatty acid binding protein. Examples of an extremely X-ray-resistant protein

Fatty acid binding protein (pI 7.0) from bovine liver cytosol was crystallized using polyethylene glycol 4000 and 6000 as precipitating agents. The crystals are triclinic, space group P1. One molecule of 14 kDa occupies the unit cell with constants a = 33.5 A, b = 39.4 A, c = 30.6 A, alpha = 113.6 degrees, beta = 113.8 degrees, gamma = 88.8 degrees. Crystal diffraction extends to at least 2.25 A resolution and the crystals are stable in the X-ray beam for more than 450 h. One native data set to 2.5 A resolution has been collected.     

Purification and crystallization of Lactobacillus casei folylpolyglutamate synthetase expressed in Escherichia coli.

Folylpolyglutamate synthetase (FPGS) from Lactobacillus casei has been crystallized with polyethylene glycol and acetate buffer at pH 5.0. The enzyme was obtained from Escherichia coli strain SF4 harboring the L. casei FPGS chromosomal gene on a pEMBL vector (pGT3-8.1). Crystals of the enzyme were obtained which diffract to 2.6 A resolution. The crystals are monoclinic, space group P2(1), with unit cell dimensions of a = 54.07 A, b = 45.83 A, c = 84.37 A and beta = 107.92 degrees. A unit cell contains one molecule of the 43,000 Da enzyme per asymmetric unit. A complete X-ray data set on the native crystals has been collected.     

Characterization of mouse tumor cell beta-lipotropin.

Mouse tumor cell beta-lipotropin (beta LPH) and gamma-lipotropin (gamma LPH) were purified from mouse pituitary tumor cell culture medium by ion exchange chromatography and gel filtration. The mouse tumor cell beta LPH was identified by immunoprecipitation with several antisera to beta-endorphin, generation of opioid bioactivity upon brief treatment with trypsin, and its identity with the molecule previously shown to serve as an intermediate in the biosynthesis of beta-endorphin. Mouse tumor cell beta LPH (Mr = 8200 +/- 250) and gamma LPH (Mr = 4600 +/- 200) are significantly smaller than known mammalian beta LPH (Mr = 10,000) and gamma LPH (Mr = 6300) molecules. The beta-endorphin region of mouse tumor cell beta LPH has the same amino acid composition as ovine, bovine, and camel beta-endorphin, and species-specific differences are thus located in the gamma LPH region of the molecule. Mouse tumor cell beta LPH and gamma LPH lack a methionine residue at what had been considered to be a highly conserved site in their beta-melanotropin-like region. A species-specific radioimmunoassay for mouse tumor cell gamma LPH was developed. Rat pituitary beta LPH and gamma LPH were shown to be similar to the corresponding mouse tumor cell molecules in size and lack of methionine in their beta-melanotropin-like segment.     

Crystallization of rat intestinal fatty acid binding protein. Preliminary X-ray data obtained from protein expressed in Escherichia coli

Rat intestinal fatty acid binding protein has been expressed in Escherichia coli, purified with bound long chain fatty acids and crystals grown from solutions of polyethylene glycol 4000. The crystals are monoclinic, space group P2(1), a = 3638 A, b = 57.2 A, c = 31.9 A, and beta = 113.9 degrees. Each unit cell contains two monomers of this 132-residue, 15.1-kDa polypeptide. The crystals are remarkably resistant to x-ray damage. X-ray diffraction data have been observed to 2.0 A resolution. Platinum chloride was used to generate a potential isomorphous heavy atom derivative.