Crystallization and preliminary X-ray diffraction analysis of oucleoside diphosphate kinase from Myxococcus xanthus.

Nucleoside diphosphate (NDP) kinase catalyzes the transfer of the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. Human and rodent forms of this enzyme have been shown to be suppressors of metastasis. Crystals that diffract X-rays to high resolution have been obtained for the recombinant Myxococcus xanthus NDP kinase expressed in and purified from Escherichia coli. Two crystal forms have been obtained. Both forms are orthorhombic, space group I222 (or I2(1)2(1)2(1)) with a = 267.1 A, b = 74.0 A and c = 75.1 A for form I and a = 53.5 A, b = 74.0 A and c = 75.1 A for form II. Form I appears to have five molecules in the asymmetric unit approximately related to each other by a translation of 0.2 along the a axis. Diffraction data have been recorded to 1.9 A for form I and to 2.2 A for form II.     

Crystallization and preliminary analysis of crystals of cytochrome c2 from Rhodopseudomonas capsulata

Two crystal forms of the cytochrome c2 isolated from Rhodopseudomonas capsulata have been obtained. One crystal form (type I), grown from ammonium sulfate solutions at pH 7.5, belongs to the space group R32 with unit cell dimensions of a = b = 100.0 A, and c = 162.2 A in the hexagonal setting. These crystals most likely contain two molecules in the asymmetric unit. The other crystal form (type II) was obtained from polyethylene glycol 6000 solutions at pH 6.5. Type II crystals belong to the space group P3(1)21 or P3(2)21 with one molecule per asymmetric unit and unit cell dimensions of a = b = 52.4 A, and c = 87.9 A. Both crystal forms diffract to at least 1.8 A resolution and appear to be resistant to radiation damage.     

Crystallization and preliminary X-ray analysis of the lactose-specific phosphocarrier protein IIAlac of the phosphoenolpyruvate: sugar phosphotransferase system from Staphylococcus aureus.

The IIA constituent of the lactose permease from Staphylococcus aureus has been crystallized in two different forms. Crystals of form I have been grown from polyethylene glycol 4000 with beta-octyl glucoside. They diffract to 3.0 A resolution and belong to space group C2 with unit cell dimensions a = 141.7 A, b = 130.7 A, c = 96.5 A and beta = 96.2 degrees. Form II crystals have been obtained from a solution containing polyethylene glycol 400, ammonium sulfate and manganese chloride. They diffract to at least 2.8 A resolution and belong to space group P2(1)2(1)2(1) with unit cell dimensions a = 89.9 A, b = 101.5 A and c = 90.9 A.     

Crystallization of a complex between ribonuclease Ms and 3'-guanylic acid

The crystals of a complex between ribonuclease Ms, the extracellular ribonuclease from Aspergillus saitoi, and 3'-guanylic acid were obtained from 2-methyl-2,4-pentanediol solution by vapor diffusion technique in the hanging drop mode. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with dimensions a = 47.0 A, b = 62.8 A, c = 37.9 A. The crystals diffract strongly up to at least 2.0 A resolution.     

Preliminary X-ray study of crystals of human C-reactive protein

Two different crystal forms of human C-reactive protein have been grown from solutions of 2-methyl-2,4-pentanediol. Both crystal forms are tetragonal, the space group for form I is P4(1)22 (or P4(3)22), and that for form II is P4(2)22. The unit cell parameters for form I are a = b = 103.0(5) A, c = 308.5(7) A and for form II are a = b = 103.1(2) A, c = 312.7(6) A. The crystals of form II diffract to at least 3.0 A resolution, and are suitable for detailed structural studies.     

Purification and crystallization of the EcoRV restriction endonuclease

The type II restriction endonuclease EcoRV purified from a genetically engineered, overproducing strain has been crystallized. Four crystal forms all obtained by precipitation with polyethylene glycol 4000 have been characterized. Two of these are suitable for high resolution structure analysis. Both are orthorhombic, have space group P2(1)2(1)2(1) and have similar unit cell dimensions of a = 58.2 A, b = 71.7 A, c = 130.6 A (form A) and a = 59.9 A, b = 74.5 A, c = 121.8 A (form B). They diffract to about 2A resolution and appear to have one dimer of 2 X 29,000 daltons in the asymmetric unit.     

Preliminary X-ray diffraction studies of the putative catalytic domain of gamma delta resolvase from Escherichia coli

Two crystal forms of the putative catalytic domain (residues 1-140) of gamma delta resolvase from Escherichia coli have been obtained. Type I is isomorphous with crystals of the intact protein, and type II is suitable for high resolution structure analysis. Type II crystals belong to the orthorhombic space group C222(1), with a = 76.8 A, b = 191.3 A, and c = 63.4 A. They contain two molecules (15,500 daltons each)/asymmetric unit and show diffraction beyond 2.7-A resolution. Calculation of a rotation function using 7-A data shows the orientation of the noncrystallographic axes.     

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 diffraction analysis of a plant ribonuclease from the seeds of the bitter gourd Momordica charantia.

Single crystals of ribonuclease Mc, a new class of plant ribonuclease from the seeds of the bitter gourd, were obtained from solutions of polyethylene glycol 8000 by the hanging-drop vapour diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 67.28 A, b = 75.21 A, c = 38.54 A. The assumption of one monomer per asymmetric unit gives rise to a Vm value of 2.29 A3/Da. The crystals diffract beyond 2.0 A resolution and are suitable for high resolution X-ray structure analysis.     

Crystallization and preliminary X-ray crystallographic studies of benzamidine-inhibited trypsin from the North Atlantic salmon (Salmo salar)

Crystals of benzamidine-inhibited trypsin from the North Atlantic salmon (Salmo salar) have been grown from ammonium sulphate solution at pH 5.0. Two crystal forms suitable for X-ray structure analysis, obtained from a hanging-drop experiment, have been characterized. Both belong to space-group P22(1)2(1) with cell dimensions a = 39.2 A, b = 62.4 A, c = 84.6 A and a = 31.4 A, b = 74.8 A, c = 83.5 A, for forms I and II, respectively. Intensity data to 1.82 A have been collected for crystal form I on a CAD4 diffractometer, and initial phases have been obtained by molecular replacement methods. The conventional R-factor after two rounds of model building and subsequent refinement is 0.25 for data between 6.0 and 2.0 A. So far no water molecules have been included in the model.     

Purification, crystallisation and preliminary X-ray study of haemoglobin from Crocodilis palustris and Crocodilis porosus

The unsolved three-dimensional structure of crocodile haemoglobin and its prospects as a blood substitute have led us to initiate the purification and crystallisation of haemoglobin molecules from crocodile species (Crocodilis palustris or mugger and Crocodilis porosus or salt water crocodile). The work has resulted in the prevention of polymerisation of naked haemoglobin molecules using N-ethylmaleimide or iodoacetamide. The purified monomeric haemoglobin molecule of C. porosus was crystallised in two different forms and X-ray diffraction data were collected up to 2 A resolution for both forms. Form I: a=53.62, b=53.55, c=103.77 A; beta=93.35 degrees, space group P2(1), Z=2. Form II: a=71.30, b=54.70, c=80.00 A; beta=106.4 degrees, space group P2(1), Z=2. Structure solution and rigid body refinement of form I data resulted in a model with R(free)=0.42 and R=0.35.     

Crystallization and preliminary analysis of the deoxyoligonucleotide d(CGTAGATCTACG)

Two crystal forms of the self-complementary DNA 12-mer d(CGTAGATCTACG) were grown by the vapour diffusion technique. Form I is in space group C2 with a = 64.8 A, b = 35.4 A, c = 24.4 A and beta = 92.2 (1 A = 0.1 nm). The crystals are grown as monoclinic blocks or hexagonal plates. There are two strands (one duplex) in the asymmetric unit. Form II crystallizes as monoclinic blocks, space group P21 with a = 64.5 A, b = 35.1 A, c = 25.2 A and beta = 91.8 degrees. This form contains four strands (2 duplexes) in the asymmetric unit. Both forms are suitable for high resolution X-ray analysis. The diffraction patterns suggest that the DNA is in a B-type conformation and that the packing in the two forms is very similar.     

Crystallization of and preliminary crystallographic data for allosteric L-lactate dehydrogenase from Bifidobacterium longum

L-Lactate dehydrogenase from Bifidobacterium longum aM101-2 was overexpressed in Escherichia coli and then purified. The enzyme was crystallized from a polyethylene glycol 6000 solution by the hanging drop vapor diffusion method. Crystals grown in the presence of NADH (type II), both NADH and oxamate (type III), and NADH, oxamate, and FBP (type IV) were analyzed. All three crystal forms belong to the orthorhombic system, space group P2(1)2(1)2. The cell dimensions of the type II crystals were a = 106.2 A, b = 131.6 A, and c = 63.8 A. Those of the type III and type IV crystals were a = 106.4 A, b = 131.4 A, and c = 63.8 A. The type III crystals diffract X-rays to beyond 2.5 A spacing. The type II and type III crystals were stable as to X-ray irradiation.     

Crystallization and preliminary X-ray analysis of a fungal endoglucanase I.

An endoglucanase I (EG1) from a fungal source (Humicola insolens) has been crystallized in a number of forms suitable for X-ray diffraction analysis. Four crystal forms have been grown from various precipitants using vapour phase diffusion methods in hanging drops. Three of these crystal forms diffract to beyond 2.5 A resolution. Two forms, obtained from ammonium sulphate at pH 5.4, or 8.0, grow as tetragonal bipyramids in space group P4(1)22 or P4(3)22, with approximate cell dimensions a = b = 102 A, c = 282 A. The other crystal forms were grown from polyethylene glycol 8000 at pH 8.0. One grows as monoclinic plates, space group P2(1), with cell dimensions a = 66.9 A, b = 75.2 A, c = 86.9 A and beta = 102.9 degrees and the other as long hexagonal rods in space group P6(1)22 or P6(5)22, with cell dimensions a = b = 119 A, c = 83 A.     

Preliminary crystallographic analysis of the breakage-reunion domain of the Escherichia coli DNA gyrase A protein

The 64 x 10(3) Mr N-terminal breakage-reunion domain of the Escherichia coli DNA gyrase A protein was purified from an over-expressing strain. When complexed with the gyrase B protein, this truncated A protein has all of the enzymic properties of the full-length counterpart, although with reduced efficiency in some cases. The 64 x 10(3) Mr protein has been crystallized in several forms, a number of which were too small for crystallographic analysis. However, two forms grew to sufficient size for preliminary X-ray analysis. Both forms were tetragonal with a primitive lattice. One form (type I) had cell dimensions of a = b = 170 A, c = 145 A a space group of either P41212 (P43212) or P42212, and diffracted to 6 A resolution. The type II crystals had cell dimensions of a = b = 177 A, c = 175 A, a space group of P41212 (P43212) or P42212, and diffracted to at least 4.5 A resolution. Both crystal forms apparently contained four subunits (possibly a tetramer) in the asymmetric unit. We are attempting to increase the size and quality of these crystals.     

X-ray grade crystals of the enzymatic fragment of diphtheria toxin

The enzymatic fragment of diphtheria toxin, fragment A (Mr = 21,167), complexed to the dinucleotide adenosine 3',5'-uridine (ApU), has been crystallized at two different values of pH by hanging drop vapor diffusion. Crystals grown at a pH value of 5.0 (from I) belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 71.2 A, b = 73.0 A, c = 139.8 A and four protomers in the asymmetric unit. Crystals grown at a pH value of 8.1 (form II) belong to the monoclinic space group C2, with unit cell parameters a = 65.2 A, b = 85.6 A, c = 34.6 A, beta = 103.0 degrees and one protomer in the asymmetric unit. Both crystal forms diffract to 2.5 A resolution. The molecular structures of fragment A obtained from these two crystal forms may illuminate the pH-dependent transition of diphtheria toxin during membrane translocation.     

New crystal forms and low resolution structure analysis of 20S proteasomes from bovine liver

20S proteasomes from higher eukaryotes have immunological functions rather than those from archibacteria or yeast. To clarify the mechanism of the sorting and production of antigen-presenting peptides, it is important and worthwhile to determine the structure of mammalian proteasomes using a third generation synchrotron radiation source. Here we report new crystal forms of 20S proteasomes from bovine liver and preliminary structure analysis of them. The crystals belong to the same space group but have different cell dimensions. One crystal (form I) belongs to space group P2(1)2(1)2(1) with unit cell dimensions of a = 124.8, b =197.4, c =323.8 A, and diffracts to 3.0 A resolution. The other crystal (form II) belongs to the same space group with a =115.1, b =205.6, c =316. 0 A, and diffracts to 4.0 A resolution. The diffraction data for the form I crystal provided an interpretable electron density map for presenting the structural differences from yeast proteasomes.     

Crystallization, X-ray diffraction analysis and phasing of carboxylesterase PA3859 from Pseudomonas aeruginosa

We have recently purified an intracellular carboxylesterase encoded by the open reading frame PA3859 of Pseudomonas aeruginosa. Among proteins showing a significant sequence homology with PA3859 the in vivo function is only known for the human acyl-protein thioesterase I that is involved in the deacylation of Galpha proteins. The crystal structure determination of P. aeruginosa carboxylesterase is expected to provide insights into its physiological role. Therefore, the PA3859 gene was cloned and heterologously expressed in Escherichia coli as N-terminally 6xHis tagged recombinant protein. Here, we present the crystallization, X-ray diffraction analysis and phasing of this enzyme. Two crystal forms were obtained by the hanging drop vapor diffusion method. Crystals of form I belong to the space group P2(1) with cell dimensions of a=65.65, b=50.55, c=142.55 A, beta=92.9 degrees and diffracted, upon flash annealing, up to a resolution of 2.9 A. Two dimers are present in the asymmetric unit. Crystals of form II belong to space group P2(1)2(1)2, with unit cell dimensions of a=96.42, b=96.36, c=68.04 A and diffracted up to 2.1 A resolution. One dimer is present in the asymmetric unit.     

Crystallization and preliminary X-ray diffraction studies of the Lb proteinase from foot-and-mouth disease virus.

Different crystal forms of the C23A mutant from the leader proteinase of foot-and-mouth disease virus were obtained by the hanging drop vapor diffusion technique, using MgCl2 and PEG 6000 as precipitants. Well-developed crystals, with cubic morphology growing to approximately 1.0 mm3 in size, presented a large unit cell parameter of 274.5 A and diffracted to, at most, 5 A resolution. A second type of crystal had a tetragonal appearance and these were obtained in droplets soaked in a silica gel matrix. These crystals, with an approximate size of 0.3 X 0.3 X 0.7 mm3, diffracted to approximately 4.0 A resolution, but presented a strong anisotropic mosaicity around the longest crystal axis. Crystals with a needlelike morphology and reaching sizes of about 0.2 X 0.3 X 1.2 mm3 diffracted beyond 3.5 A resolution and were stable to X-ray radiation for approximately one day when using a conventional source at room temperature. These crystals are orthorhombic with space group I222 (or I2(1)2(1)2(1)) and unit cell dimensions a = 65.9 A, b = 104.3 A, and c = 124.0 A, and appear well suited for high-resolution studies. Density packing considerations are consistent with the presence of two molecules in the asymmetric unit and a solvent content of approximately 54%.     

Characterization of two crystal forms of human defensin neutrophil cationic peptide 1, a naturally occurring antimicrobial peptide of leukocytes

Two orthorhombic crystal forms (P2(1)2(1)2, a = 30.5 A, b = 44.5 A, c = 40.5 A; I2(1)2(1)2(1) (or I222), a = 30.1 A, b = 66.5 A, c = 35.5 A) of human neutrophil cationic peptide 1 have been characterized. The P2(1)2(1)2 form contains two peptides (Mr = 3425) per asymmetric unit; the I2(1)2(1)2(1) form contains one peptide per asymmetric unit. Both crystal forms diffract to beyond 1.8 A resolution.