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 X-ray studies of the pectate lyase from Bacillus subtilis.

The pectate lyase (EC 4.2.2.9) from Bacillus subtilis has been crystallized. Crystals of form 1, grown by the hanging drop method using polyethylene glycol as precipitant, diffract to at least 2.4 A resolution. They belong to the spacegroup P2(1) with a = 132.9 A, b = 41.2 A, c = 156.8 A and beta = 114.9 degrees with probably four molecules in the asymmetric unit. A second crystal form grown from 2-methyl-2,4-pentandiol also belongs to the spacegroup P2(1) with a = 55.0 A, b = 88.1 A, c = 50.2 A and beta = 109.0 degrees. These crystals diffract to at least 2.0 A and have one molecule in the asymmetric unit. Both crystal forms are suitable for the determination of high-resolution structures.     

Crystallization and preliminary X-ray study of pig lens aldose reductase

Crystals of pig lens aldose reductase have been grown from polyethylene glycol solutions at pH 6.2 and analysed by X-ray diffraction. Two crystal forms were obtained. The first belongs to space group P1 with unit cell dimensions a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees, gamma = 79.0 degrees, with four molecules in the unit cell related by a 222 non-crystallographic symmetry. The second crystal form is hexagonal. The space group is P6(2)22 with a = b = 101 A, c = 257 A and two molecules in the asymmetric unit. Both forms are suitable for X-ray structure analysis to better than 3 A resolution.     

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 and preliminary diffraction studies of hydroxypyruvate reductase (D-glycerate dehydrogenase) from Hyphomicrobium methylovorum.

Two crystal forms of hydroxypyruvate reductase (D-glycerate dehydrogenase) from the methylotrophic bacterium Hyphomicrobium methylovorum have been grown from ammonium sulphate solutions. One crystal form is triclinic, with unit cell parameters a = 60.4 A, b = 60.5 A, c = 66.3 A, alpha = 102.3 degrees, beta = 113.7 degrees and gamma = 102.7 degrees, suggesting that a dimer (monomer M(r) 38,000) occupies the unit cell. This crystal form diffracts to beyond 2.4 A resolution and is suitable for crystallographic structure analysis.     

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.     

Two crystal forms of Azotobacter ferredoxin.

Two crystal forms of Azotobacter vinelandii (4Fe-4s)2 ferredoxin I (Fd I) have been grown which are suitable for high resolution x-ray diffraction studies. Tetragonal crystals grow as square bipyramids from ammonium sulfate and Tris buffer using a temperature gradient. The space group is P41212 (or P43212) with a = 55.3, c = 95.9 A and 1 molecule/asymmetric unit. Triclinic crystals grow as plates or laths from ammonium sulfate and phosphate buffer at constant temperature. The space group is P1 with a = 46.8, b = 58.7, c = 64.3 A, alpha = = 105 degrees 05 min, beta = 82 degrees 30 min, gamma = 110 degrees 30 min and 4 or 5 molecules/unit cell. Both crystal forms are stable to x-ray irradiation and diffract beyond 3.0 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.     

Preparation, crystallization and preliminary X-ray analysis of the Fab fragment of monoclonal antibody MN423, revealing the structural aspects of Alzheimer's paired helical filaments

Monoclonal antibody (mAb) MN423 recognizes Alzheimer's disease specific conformation of tau protein assembled into paired helical filaments (PHF). Since the three-dimensional structure of PHF is currently unavailable, the structure of MN423 binding site could provide important information about PHF conformation with the consequences for the Alzheimer's disease prevention and cure. Fab fragment of MN423 was prepared and purified. We have identified two different conditions for crystallization of the Fab fragment that yielded two crystal forms. They diffracted to 3.0 and 1.6 A resolution with four and one molecule in the asymmetric unit, respectively. Both crystal forms belonged to the space group P2(1) with unit cell parameters a = 76.4 A, b = 138.4 A, c = 92.4 A, beta = 101.9 degrees , and a = 71.5 A, b = 36.8 A, c = 85.5 A, beta = 113.9 degrees .     

Crystallization and characterization of two crystal forms of the B800-850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

Two different crystal forms of the B800-850-antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown. This complex is an integral membrane protein and is isolated as an oligomeric assembly with a molecular weight of approximately 84 kDa. This assembly contains six alpha/beta apoprotein pairs, 18 molecules of bacteriochlorophyll a and nine molecules of carotenoid. The first crystal form has dimensions unit cell a = b = 75.8 A, c = 97.5 A with the space group P4 and diffracts to a resolution of 12.0 A. The second crystal form is rhombohedral with dimensions unit cell a = 121.1 A, alpha = 60 degrees, space group R32 and diffracts to a resolution of 3.5 A. Native data have been processes in both cases, to an Rmerge value of 9.0 to 11.0%. The X-ray data suggest that the asymmetric unit, in both crystal forms, contains one 84 kDa antenna complex.     

Crystallization and preliminary X-ray crystallographic analysis of the 38-kDa immunodominant antigen of Mycobacterium tuberculosis.

The 38-kDa lipoprotein is one of the most potent cell surface immunogens of Mycobacterium tuberculosis in antibody-and T cell-mediated reactions. Using a pure recombinant form of the protein, we have recently shown that it binds phosphate much like that of the phosphate-binding protein (M(r) = 34.4 kDa) that is localized in the periplasm of Escherichia coli and is involved as an initial receptor for active transport of phosphate. The purified 38-kDa protein has been crystallized in 2 forms that are suitable for high-resolution structural analyses. One form belongs to the monoclinic space group P2(1) with unit cell dimensions of a = 67.42 A, b = 113.38 A, c = 42.68 A, and beta = 108.53 degrees. The other is of orthorhombic space group P2(1)2(1)2 with a = 125.46 A, b = 72.27 A, and c = 73.43 A. Both crystal forms diffract to about 2 A resolution on a fine focus rotating anode.     

Crystallization and preliminary diffraction studies of recombinant human granulocyte-stimulating factor (KW2228).

Human granulocyte colony-stimulating factor (hG-CSF) specifically stimulates proliferation of neutrophils. Two crystal forms of a mutant of hG-CSF expressed in Escherichia coli have been obtained using the hanging drop vapour diffusion method. One form is triclinic, space group P1, with cell dimensions a = 37.3 A, b = 46.4 A, c = 47.7 A, alpha = 105.5 degrees, beta = 98.0 degrees and gamma = 109.4 degrees. The other is monoclinic, space group C2, with cell dimensions a = 82.0 A, b = 49.2 A, c = 49.4 A and beta = 113.9 degrees. Both crystal forms diffract beyond 2.0 A and are suitable for X-ray analysis.     

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.     

Human plasminogen kringle 4. Crystallization and preliminary diffraction data of two different crystal forms

Human plasminogen kringle 4 has been crystallized in two different crystal forms: monoclinic, a = 32.78(3), b = 49.17(2), c = 46.27(3) A, beta = 100.67 degrees, space group P2(1), four molecules/unit cell, two molecules/asymmetric unit; orthorhombic, a = 32.09(7), b = 49.14(6), c = 49.47(9) A, space group P2(1)2(1)2, four molecules/unit cell. Both crystal forms have a large protein fraction (66% for monoclinic and 62% for orthorhombic) and diffract x-rays to 2.0 A resolution. A self-rotation function has been calculated with monoclinic data indicating a non-crystallographic 2-fold rotation approximately parallel to a* (peak height of 14.3 x sigma). Cross-rotation function calculations are in progress utilizing the coordinates of the conserved structure of kringle 1 of prothrombin and plasminogen kringle 4.     

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.     

Preparation and X-ray characterization of four new crystal forms of jacalin, a lectin from Artocarpus integrifolia

Four new crystal forms of the anti-T lectin from jackfruit (Artocarpus integrifolia) have been prepared and characterized. Three of them, two monoclinic (P21, a = 59.4 A, b = 83.3 A, c = 63.5 A, beta = 107.7 degrees; C2, a = 106.1 A, b = 53.9 A, c = 128.0 A, beta = 95.0 A) and one orthorhombic (C222(1), a = 98.1 A, b = 67.3 A, c = 95.1 A) were grown with 2-methylpentan-2,4-diol (MPD) as the precipitant while the fourth, an hexagonal form (P6(1)22, a = b = 129.6 A, c = 157.9 A), was obtained in the presence of methyl-alpha-D-galactopyranoside with polyethylene glycol 4000 as the precipitant. The reported relative molecular mass (Mr) of the lectin was found to be inconsistent with the solvent content of the crystals estimated using measured densities. The Mr was redetermined using size-exclusion chromatography in the presence of methyl-alpha-D-galactopyranoside and Ferguson-plot analysis of mobilities in polyacrylamide gel electrophoresis. The redetermined Mr (66,000) is consistent with the measured crystal densities. The orthorhombic and the hexagonal forms, which have one half molecule and one molecule, respectively, in the asymmetric unit, are suitable for high-resolution X-ray analysis.     

Crystallization of and preliminary X-ray data for the mouse major urinary protein and rat alpha-2u globulin.

Crystals of the mouse major urinary protein (MUP) and rat alpha-2u globulin (AMG) have been grown from solutions of polyethylene glycol 3350 and CdCl2, respectively. The crystals differ both in their morphologies and space groups but have very similar unit cell sizes. AMG crystallized in P2(1) (a = 56.6 A, b = 103.8 A, c = 62.7 A, beta = 95.1 degrees) with four subunits/asymmetric unit, while MUP gave crystals in P4(1)2(1)2 or P4(3)2(1)2 (a = 57.3 A, c = 109.9 A) with one subunit/asymmetric unit. Both crystal forms diffract beyond 2.8 A resolution.     

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 crystal data of porcine pepsinogen

Single crystals of porcine pepsinogen, suitable for x-ray diffraction studies, have been grown with lithium sulfate as the precipitant. These pepsinogen crystals were dissolved, activated, and assayed for proteolytic activity. The specific enzymic activity of the dissolved crystalline protein was nearly twice that of the commerical pepsinogen from which the crystals were grown. Incubation at pH 8 before assay demonstrated that the crystals are free of pepsin. This crystal form of pepsinogen belongs to the monoclinic space group C2 with 4 molecules in the unit cell. The unit cell dimensions are a = 104.8 +/- 0.5 A, b = 43.1 +/- 0.1 A, c = 88.4 +/- 0.3 A, and beta = 91.3 degrees.     

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.