Crystallization and preliminary X-ray crystallographic analysis of verotoxin-1 B-subunit

The B-subunit of verotoxin-1, which is believed to form a pentamer (monomer Mr = 7691), has been crystallized by vapor diffusion over a wide range of conditions. The best crystals, obtained with polyethylene glycol 8000 as the precipitant, belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 59.2 A, b = 102.7 A, c = 56.3 A. The cell dimensions are consistent with one B-subunit pentamer per asymmetric unit, and the crystals diffract to at least 2.0 A resolution. Data collected using synchrotron radiation at a wavelength of 2.070 A may allow the structure to be solved using the anomalous signal from three sulfur atoms in the monomer, combined with averaging over the non-crystallographic symmetry.     

A 9 A two-dimensional projected structure of cholera toxin B-subunit-GM1 complexes determined by electron crystallography.

Highly ordered two-dimensional crystals of cholera toxin B-subunit pentamers have been grown by specific interaction with planar lipid films containing monosialoganglioside GM1. Electron diffractograms of frozen-hydrated crystals show diffraction peaks extending to beyond 4 A, while electron images diffract to 8 A. A two-dimensional projected structure of cholera toxin B-subunit-GM1 complex has been calculated at 9 A resolution by combining electron diffraction and image data. Crystals present an approximate pgg projection symmetry, with unit cell dimensions a = 119(+/- 1) A, b = 123(+/- 1) A, gamma = 90 degrees. Each pentameric assembly presents two concentric rings of electron scattering density, separated by an area of lower density. The outer and inner rings are centered at 25 A and and 11 A from the pentamer centre, respectively. The apparent projected density of the outer ring is larger than that of the inner ring. We propose that the outer and inner density rings correspond respectively to the peripheral beta-sheet arrangement and the central alpha-helix barrel, recently identified in the crystal structure of the heat-labile enterotoxin from Escherichia coli.     

Crystallization and preliminary x-ray diffraction study of synthetic apolipoprotein E fragment (residues 129-169)

Apolipoprotein E is a plasma protein comprised of a lipid binding region (which together with other apoproteins maintains the structure of lipoprotein particles) and a receptor binding domain (which interacts with cellular receptors for control of triglyceride and cholesterol metabolism). A peptide, comprising residues 129-169 of human apolipoprotein E, which contains both a putative lipid-binding region and receptor binding domain, has been synthesized by solid phase techniques. Diffraction quality crystals of the synthetic apolipoprotein E fragment129-169 have been obtained at room temperature by vapor diffusion with polyethylene glycol in the presence of the nonionic detergent beta-octylglucoside. The crystals have been characterized with x-radiation as orthorhombic, space group I222 or I2(1)2(1)2(1), with unit cell dimensions a = 61.91, b = 30.84, and c = 42.79 A. There are eight molecules per unit cell, with one molecule (Mr = 4771) in each asymmetric unit. Precession photographs show that crystals diffract beyond 2.7-A resolution and are stable in the x-ray beam at room temperature for at least 200 h; thus, they can be used to collect three-dimensional data for a detailed crystallographic analysis.     

Crystallization of serine carboxypeptidases

Crystallization of three different serine carboxypeptidases has been achieved by the method of hanging-drop vapor diffusion. Serine carboxypeptidases II from wheat bran and malted barley crystallize isomorphously from polyethylene glycol solutions at room temperature (pH 4 to 7) in space group P4(1)2(1)2 or enantiomorph with cell dimensions of a = b = 98.2 A and c = 209.5 A. The crystals diffract to about 2.3 A resolution using rotating-anode X-ray generators. Assuming a dimer of Mr 120,000 in the asymmetric unit, Vm = 2.1 A3/dalton. These crystals appear suitable for structural studies. A genetically engineered serine carboxypeptidase from yeast, which lacks three of four glycosylation sites present in the wild-type, has also been crystallized by vapor diffusion against methylpentanediol at 4 degrees C, pH 6.4 to 8.0.     

Crystallization and preliminary X-ray diffraction analysis of staphylococcal enterotoxin type C.

The Type C staphylococcal enterotoxin produced by Staphylococcus aureus strain FRI-909 has been crystallized using a combination of two precipitants, ammonium sulfate and polyethylene glycol 400, with the addition of small amounts of detergent. Two related crystal forms have been obtained, one triclinic, and one tetragonal, both with one toxin molecule per asymmetric unit. These crystals are stable for at least 75 hr in the X-ray beam and diffract to at least 2.2 and 2.6 A, respectively. The triclinic crystals have unit cell parameters a = 38.5 A, b = 43.7 A, c = 36.9 A, and interaxial angles alpha = 99.9 degrees, beta = 95.8 degrees, and gamma = 98.5 degrees. The tetragonal crystals are of space group P4(1)22 with unit cell parameters a = 43.4 A and c = 278.0 A.     

A crystal form of ribulose-1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum in the activated state

A new crystal form of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from Nicotiana tabacum has been obtained at alkaline pH with polyethylene glycol 8000 in the presence of a non-ionic detergent, beta-octyl glucoside. The crystals are grown at room temperature by the hanging-drop vapor diffusion technique from a protein solution containing enzyme complexed with CO2, Mg2+, and the transition state analog 2-C-carboxy-D-arabinitol-1,5-bisphosphate. The crystals belong to the the space group P3(1)21 (or P3(2)21) with the cell parameters a = 204.6 A, and c = 117.4 A (1 A = 0.1 nm). The asymmetric unit contains half (L4S4: L, large subunit, 53,000 Mr; S, small subunit, 15,000 Mr) of a hexadecameric molecule (L8S8, 540,000 Mr). The crystals diffract to at least 2.6 A Bragg spacing and are suitable for X-ray structure determination.     

Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Vibrio mimicus.

Single crystals of arylesterase (EC 3.1.1.2) from Vibrio mimicus have been obtained from ammonium sulfate as a precipitant at room temperature for 2 months. The present crystals diffract up to 2.2 A resolution and belong to monoclinic space group P2(1). The cell dimensions are a = 55.65(1) A, b = 53.46(1) A, c = 65.79(1) A, and beta = 106.54(1) degrees. There are two molecules of molecular weight 22 kDa in an asymmetric unit with a solvent content of 43%.     

Crystallization and preliminary crystallographic studies of the FAD domain of corn NADH: nitrate reductase.

Crystals of the flavin domain of corn nitrate reductase expressed in Escherichia coli have been obtained at room temperature, using sodium citrate as precipitant. The crystals diffract to at least 2.5 A resolution at a synchrotron radiation source. Precession photographs show that they belong to the rhombohedral space group R3 with unit cell dimensions a = b = 145.4 A, c = 47.5 A, alpha = beta = 90 degrees and gamma = 120 degrees. There is one subunit per asymmetric unit which gives a packing density of 3.2 A3/Da, indicating a high solvent content in these crystals.     

Crystallization of bacteriorhodopsin from bicelle formulations at room temperature

We showed previously that high-quality crystals of bacteriorhodopsin (bR) from Halobacterium salinarum can be obtained from bicelle-forming DMPC/CHAPSO mixtures at 37 degrees C. As many membrane proteins are not sufficiently stable for crystallization at this high temperature, we tested whether the bicelle method could be applied at a lower temperature. Here we show that bR can be crystallized at room temperature using two different bicelle-forming compositions: DMPC/CHAPSO and DTPC/CHAPSO. The DTPC/CHAPSO crystals grown at room temperature are essentially identical to the previous, twinned crystals: space group P21 with unit cell dimensions of a = 44.7 A, b = 108.7 A, c = 55.8 A, beta = 113.6 degrees . The room-temperature DMPC/CHAPSO crystals are untwinned, however, and belong to space group C222(1) with the following unit cell dimensions: a = 44.7 A, b = 102.5 A, c = 128.2 A. The bR protein packs into almost identical layers in the two crystal forms, but the layers stack differently. The new untwinned crystal form yielded clear density for a previously unresolved CHAPSO molecule inserted between protein subunits within the layers. The ability to grow crystals at room temperature significantly expands the applicability of bicelle crystallization.     

Crystallization and preliminary X-ray studies of two isolectins from the seeds of Lathyrus ochrus

Two isolectins from the seeds of Lathyrus ochrus, LOL I and LOL II, which specifically bind N-acetyllactosamine, have been crystallized using the hanging-drop method and the interface diffusion method, respectively. In the case of LOL I, 2-methylpentane-2,4-diol, polyethylene-glycol 400 or ammonium sulphate have been used as precipitating agents. The best crystals of LOL I were grown at room temperature from a solution of 40% (v/v) methylpentane diol, 50 mM-Hepes at pH 7.5. LOL II crystals have been grown at room temperature from a solution of 32% (v/v) methylpentane diol, 50 mM-2-(N-morpholino)-ethanesulphonic acid at pH 5.5. X-ray examination of the LOL I and LOL II crystals shows that both are monoclinic, space group P2(1). Their cell dimensions are: LOL I, a = 56.4 A, b = 138.8 A, c = 62.9 A, beta = 91 degrees; and LOL II, a = 54.8 A, b = 71.4 A, c = 105.5 A, beta = 105 degrees. Density measurements of the crystals of LOL I indicate that there are two molecules per asymetric unit (Vm = 2.07 A3/dalton). LOL I crystals diffract strongly up to at least 1.8 resolution. Putative crystals of complexes of LOL I with various glycosides were obtained through co-crystallization under the conditions used for the native protein.     

Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B

The glutathione synthetase from Escherichia coli B has been crystallized from 27% saturated ammonium sulfate solution (pH 5.5). The crystals are hexagonal, space group P6(2)22 or P6(4)22. The cell dimensions are a = b = 88.0 A, c = 164.2 A, and gamma = 120 degrees. The enzyme is a tetramer (Mr = 143,000) with 222 symmetry, and the asymmetric unit contains one subunit molecule (Mr = 35,600). The crystals diffract to at least 2.5 A resolution.     

Crystallization and preliminary X-ray analysis of pig kidney DOPA decarboxylase.

DOPA decarboxylase from pig kidney, an alpha 2 dimeric enzyme of Mr = 107,000, has been crystallized by the vapour diffusion method with ammonium sulphate as precipitant. The crystals belong to the space group P6(2) (or its enantiomer P6(4)) and have unit cell dimensions of a = b = 155.9 A, c = 87.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. They diffract to 2.6 A resolution. There is one dimeric molecule per asymmetric unit. Rotation function studies have revealed the orientation of the non-crystallographic 2-fold axis of the dimer in the asymmetric unit.     

Crystallization of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10

Haloalkane dehalogenases are enzymes that release chloride or bromide from n-halogenated alkanes. X-ray quality crystals of haloalkane dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 64% saturated ammonium sulfate solutions (pH 6.2 to 6.4). The crystals diffract in the X-ray beam to at least 2.4 A resolution (1 A = 0.1 nm). Their space group is P2(1)2(1)2, with cell dimensions a = 94.1 A, b = 72.8 A, c = 41.4 A and alpha = beta = gamma = 90 degrees. There is one monomer (molecular weight 36,000) per asymmetric unit.     

Crystallization and preliminary X-ray analysis of phosphoserine aminotransferase from Bacillus circulans subsp. alkalophilus.

Recombinant phosphoserine aminotransferase (EC 2.6.1.52) from Bacillus circulans subsp. alkalophilus was crystallized at room temperature from 0.1 M sodium acetate buffer, pH 4.6, and 2% PEG 20000, using macroseeding techniques. The crystals diffract X-rays to at least 2.0 A nominal resolution. They belong to space group C2 with unit cell dimensions a = 93.2 A, b = 93.1 A, c = 45.6 A, alpha = 90.0 degrees, beta = 106.8 degrees, gamma = 90.0 degrees. A native data set to 2.3 A has been collected. Assuming an average packing density of the crystals, there is one monomer in the asymmetric unit, resulting in a calculated solvent content of 48.2%.     

Incomplete factorial search for conditions leading to high quality crystals of Escherichia coli cytidine deaminase complexed to a transition state analog inhibitor

We have used an incomplete factorial design (Carter, C. W., and Carter, C. W., Jr. (1979) J. Biol. Chem. 254, 12219-12223) to find conditions for growing high quality crystals of Escherichia coli cytidine deaminase (EC 3.5.4.5). Crystals grow at pH 6.0 in hanging or sitting drops with either 1.6 M ammonium sulfate or 2.4-2.5 M sodium phosphate as precipitant. Both conditions produce crystals with identical morphologies and unit cell constants. The space group is P3(1)21 (or its enantiomorph P3(2)21), and the unit cell constants are a = b = 120.3 A, c = 78.4 A. The asymmetric unit is most reasonably one dimer of 66,000 Mr. The crystal size is very dependent on the supersaturation ratio, S = [initial protein concentration]/[equilibrium protein concentration], exhibiting a maximum at S = 7.7. The largest crystals diffract to at least 2.5 A and have a lifetime of 4 to 5 days in the x-ray beam at room temperature. The enzyme in these crystals is complexed with the transition state analog inhibitor 1-(beta-D-ribofuranosyl)-5-fluoropyrimidin-2-one (5-fluoropyrimidin-2-one riboside). We have collected data from parent crystals and from a heavy atom derivative in which the transition state analog is replaced by the active site directed inhibitor 5-(chloromercuri)cytidine.     

Crystallization and preliminary X-ray studies of Escherichia coli glycerol kinase

Escherichia coli glycerol kinase, a major regulatory enzyme which catalyzes the reversible MgATP-dependent phosphorylation of glycerol has been crystallized by the hanging drop vapor diffusion method at room temperature. Three different crystal forms have been obtained in the presence of glycerol and appear to be suitable for X-ray crystallographic studies. Vapor diffusion against 55% ammonium sulfate and 1% beta-octyl glucoside (pH 7.0) yields rhombohedral crystals with space group R32, a = b = 277.1 A, c = 78.7 A (hexagonal indexing) containing a dimer of Mr 112,000 in the asymmetric unit (Vm = 2.64 A3/dalton). Vapor diffusion against sodium chloride in the presence of 10% (w/v) polyethylene glycol (pH 6.5 to 7.0) yields two different crystal forms, both with space group P2(1). The first form has a = 88.1 A, b = 99.3 A, c = 114.6 A, beta = 119 degrees, the second form has a = 92.5 A, b = 117.6 A, c = 108.3 A, beta = 93.64 degrees. Addition of ADP enhances growth of the monoclinic forms. These forms appear to contain an entire tetramer of Mr 224,000 in the asymmetric unit and have Vm values of 2.28 and 2.65 A3/dalton, respectively. All forms diffract to better than 3.0 A resolution while the second monoclinic form diffracts to approximately 1.8 A.     

Preliminary X-ray crystallographic study of methanol dehydrogenase from Methylophilus methylotrophus

Single crystals of methanol dehydrogenase from Methylophilus methylotrophus have been prepared by the macroseeding method. The crystals belong to the monoclinic space group C2, and have unit cell parameters a = 125.62 A, b = 63.83 A, c = 83.99 A, and beta = 93.24 degrees. There is one 62,000 Mr monomer in the asymmetric unit. The crystals diffract to beyond 2.0 A resolution.     

Crystallization and preliminary X-ray crystallographic analysis of lipase from Pseudomonas cepacia.

Large crystals of lipase from Pseudomonas cepacia have been grown at room temperature from solutions containing 2-methyl-2,4-pentanediol and sodium citrate. They grow within two weeks to typical dimensions of 1.0 mm x 0.5 mm x 0.3 mm. The crystals belong to the monoclinic space group P2(1), with unit cell parameters a = 84.91 A, b = 47.33 A, c = 86.00 A, and beta = 116.09 degrees. And they diffract to about 1.6 A upon exposure to synchroton X-rays. X-ray data have been collected to 2.2 A Bragg spacing from a native crystal.     

Crystallization of trimeric recombinant human tumor necrosis factor (cachectin)

Crystals of tumor necrosis factor (TNF) have been obtained in two forms. Rhombohedral crystals grow in 1.8 to 2.0 M ammonium sulfite, pH 7.8 at 21 degrees C, and tetragonal crystals grow in 2.6 M magnesium sulfate, pH 5.5 at 25 degrees C. Analysis of TNF by isoelectric focusing under native and denaturing conditions indicates that TNF molecules exist as trimers in solution. The rhombohedral cachectin crystals belong to space group R3 and have unit cell constants a = b = c = 47.65 A and alpha = beta = gamma = 88.1 degrees. Density determinations and the space group indicate that the unit cell contains one 51,000-dalton trimer. These crystals are stable in the x-ray beam and diffract to at least 1.85 A but are apparently twinned by merohedry. The tetragonal crystals are space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2 and have unit cell constants a = b = 95.08, c = 117.49. The asymmetric unit contains one trimer; the crystals are stable in the x-ray beam and diffract to beyond 3 A.     

Crystallization of the A-domain of the mannitol transport protein enzyme IImtl.

The A-domain of the mannitol transport protein enzyme IImtl from Escherichia coli (relative molecular mass 16,300) was crystallized, both at room temperature and 4 degrees C, from 40% polyethylene glycol 6000 (pH 8.5 to 9.0) using the hanging-drop method of vapour diffusion. The crystals have the monoclinic space group P2(1), with unit cell dimensions a = 54.0 A, b = 67.0 A, c = 80.9 A and beta = 100.8 degrees. They diffract to 2.6 A resolution. A self-rotation function and self-Patterson suggest that there are four molecules in the asymmetric unit showing mmm symmetry.