Crystallization and preliminary X-ray diffraction analysis of recombinant pentalenene synthase.

Recombinant pentalenene synthase, a 42.5-kDa sesquiterpene cyclase originally isolated from Streptomyces UC5319 and cloned in Escherichia coli, has been crystallized in space group P6(3) with unit cell dimensions a = b = 183.5 A and c = 56.5 A. Hexagonal prismatic crystals, approximately 0.2 x 0.2 x 0.3 mm, diffract to approximately 2.9 A resolution using monochromatic synchrotron radiation. From the universal (and achiral) building block, farnesyl pyrophosphate, pentalenene synthase catalyzes the formation of four stereocenters in the construction of the three fused five-membered rings of pentalenene; this novel sesquiterpene is a precursor to the pentalenolactone family of antibiotics.     

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.     

Crystallization and preliminary X-ray diffraction analysis of 11 S acetylcholinesterase

The 11 S form of acetylcholinesterase from Electrophorus electricus was purified by affinity chromatography. The protein was crystallized from polyethylene glycol solutions. One crystal form proved suitable for x-ray diffraction studies. Preliminary x-ray analysis demonstrates that the space group of this crystal is F222. The unit cell dimensions are a = 141.0 +/- 0.2, b = 202.4 +/- 0.2, and c = 237.4 +/- 0.1 A. The diffraction is anisotropic, extending to at least 3.5 A along the a* and b* axes, but becoming weak beyond about 6 A along the c* axis. Crystal density measurements suggest that one complete 11 S tetramer occupies the asymmetric unit of the crystal.     

Crystallization and preliminary X-ray diffraction analysis of foot-and-mouth disease virus

Foot-and-mouth disease virus has been crystallized with the objectives of (1) determining the composition and conformation of the major immunogenic site(s) and (2) comparing its structure with those of the related polio, rhino and Mengo viruses, representing the other three genera of the picornaviruses. Most of the work has been done with virus strain O1BFS 1860, which crystallized as small rhombic dodecahedra of maximum dimension 0.3 mm. Virus recovered from crystals was infectious, and was indistinguishable from native virus both in protein composition and buoyant density. The stability of the crystals in the X-ray beam was comparable with that of other picornavirus crystals and they diffracted to a resolution of better than 2.3 A. Initial analysis of the X-ray diffraction data shows the virus to be positioned on a point of 23 symmetry in a close-packed array so that examples of all the icosahedral symmetry elements, except the 5-fold axes, are expressed crystallographically. The cell dimensions are a = b = c = 345 A, alpha = beta = gamma = 90 degrees, with a space group of I23. The diameter of the virus particle is 300 A. Despite the small size of the crystals, diffraction data have been collected to a reasonable resolution using a synchrotron source. Phasing of the diffraction data will be attempted using the methods of molecular replacement.     

Crystallization and preliminary X-ray diffraction analysis of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA Synthetase, a class I aminoacyl tRNA synthetase playing a crucial role in protein biosynthesis, has been crystallized for the first time. Polyethylene glycol (PEG) was used as a precipitant, and the crystallization proceeded at pH 6.5. These single crystals diffracted to 2.8 A with a rotating anode X-ray source and R-axis IIc image plate detector. They have an orthorhombic space group P2(1)2(1)2 with unit cell parameters of a = 251.51 A, b = 53.12 A, and c = 52.35 A. A complete native data set has been collected at 3.1 A resolution for these crystals.     

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 X-ray diffraction studies of the spinach-chloroplast thioredoxin f.

Thioredoxins are low-molecular-mass proteins that function as hydrogen carriers in DNA synthesis and in the transformation of sulfur metabolites. They also act as regulatory proteins in the light-dependent enzyme activation during photosynthesis. F-type thioredoxin from spinach chloroplasts, a monomeric protein of 113 amino acid residues, has been found to specifically activate fructose-1,6-bisphosphatase and other key enzymes of CO2 assimilation. It has been crystallized in the monoclinic system, space group P2(1) with a = 30.6 A, b = 63.1 A, c = 31.6 A and beta = 110.7 degrees. The crystals are suitable for X-ray diffraction studies.     

Crystallization and preliminary X-ray diffraction studies of coxsackievirus B1.

Preparations of coxsackievirus B1 (CVB1) derived from an infectious cDNA clone have been crystallized in multiple crystal forms. Using high intensity synchrotron radiation, an orthorhombic form of the crystals was shown to diffract X-rays to at least 2.9 A resolution. The unit cell has a primitive lattice with dimensions a = 323 A, b = 450 A, and c = 522 A. A crystallographic asymmetric unit of these CVB1 crystals probably contains an entire virus particle, implying the presence of 60-fold non-crystallographic redundancy. This CVB1 crystal form appears to be suitable for high-resolution structure determination by X-ray crystallography.     

Crystallization and preliminary X-ray diffraction analysis of importin-alpha complexed with NLS peptidomimetics

Importin-alpha is the nuclear import receptor that recognizes cargo proteins with nuclear localization sequences (NLSs). The study of NLS peptidomimetics can provide a better understanding of the requirements for the molecular recognition of cargo proteins by importin-alpha, and potentially engender a large number of applications in medicine. Importin-alpha was crystallized with a set of six NLS peptidomimetics, and X-ray diffraction data were collected in the range 2.1-2.5 A resolution. Preliminary electron density calculations show that the ligands are present in the crystals.     

Crystallization and preliminary X-ray diffraction analysis of phosphoglucose isomerase from Pyrococcus furiosus

In several euryarchaeota, phosphoglucose isomerase (PGI) activity is catalyzed by an enzyme unrelated to the well-known family of PGI enzymes found in prokaryotes, eukaryotes and some archaea. In order to understand the mechanistic differences between the two families of enzymes we have crystallized PGI from the archaeon Pyrococcus furiosus. The crystals belong to the space group P2(1) and a complete dataset extending to 1.9 A resolution has been collected.     

Crystallization and preliminary X-ray diffraction analysis of catalase HPII from Escherichia coli

Green crystals of the hexameric catalase HPII from Escherichia coli have been obtained by the hanging-drop method. The crystals belong to the monoclinic space group P2 with a = 123 A, b = 132 A, c = 93 A, beta = 112.5 degrees. There are three subunits in the asymmetric unit. The crystals diffract at least to 3.2 A resolution and are suitable for further X-ray diffraction studies.     

Crystallization and preliminary X-ray diffraction studies of pancreatic spasmolytic polypeptide.

Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapour diffusion method. The crystals belong to the space group I222 or I2(1)2(1)2(1) with cell dimensions a = 181.9 A, b = 54.5 A, c = 72.9 A. The crystals diffract to at least 2.5 A resolution and the asymmetric unit contains two molecules (Vm = 3.9 A3/Da) with a solvent content of 68% as determined by density measurements of the crystals. The self-rotation function suggests that the two molecules within the asymmetric unit are related by a 2-fold axis at either 30 degrees or 60 degrees from a in a plane perpendicular to the b axis.     

Crystallization and preliminary X-ray diffraction studies of human salivary alpha-amylase.

Nonglycosylated alpha-amylase, a major component of human parotid saliva, has been crystallized by the vapor diffusion technique using 2-methyl-2,4-pentanediol as the precipitant in the presence of CaCl2 at pH 9.0. The crystals are orthorhombic, space group P2(1)2(1)2(1) with unit cell dimensions of a = 53.3, b = 75.8, and c = 138.1 A. The asymmetric unit contains one amylase molecule. The solvent content is 54%. The crystals are stable to X-rays and diffract up to 2.8 A and appear to be suitable for X-ray diffraction studies.     

Crystallization and preliminary X-ray diffraction studies of soybean agglutinin

Soybean agglutinin crystallizes in the monoclinic space group C2 with unit cell dimensions $\text{a = 118.6}\text{A}\text{?}\text{, b = 88.9}\text{A}\text{?}\text{, c = 165.9}\text{A}\text{?}\text{, β = 103.0 °}$ and one tetramer of 120,000 M_r per asymmetric unit. The crystals are suitable for high-resolution work.     

Crystallization and preliminary X-ray diffraction studies of antigen--antibody complexes

Monoclonal antibodies of predefined specificity have been purified and crystallized as single components or complexed with their specific antigens. The intersegmental flexibility of antibody molecules has imposed the strategy of attempting to crystallize their Fab fragments separately. Intrasegmental mobility in Fabs has rarely been an obstacle to their crystallization. The immune system, however, provides a large functional and structural diversity of antibody molecules suitable for crystallization and X-ray diffraction studies.     

Crystallization and preliminary X-ray diffraction studies of Streptomyces phospholipase A2 in a calcium-binding form

Phospholipase A2 (PLA2) as a calcium-binding form, produced by Streptomyces violaceoruber, was crystallized in a form suitable for the diffraction analysis using the vapor diffusion method. Crystals were grown in 0.1 M Tris-HCl buffer (pH 8.5), 20 mM Ca2+ containing 50-60% (v/v) 2-methyl-2,4-pentanediol as a precipitant. They belong to the monoclinic space group P2(1), with the cell dimensions a=38.3 A, b=54.3 A, c=30.6 A, and beta=90.2 degrees. The crystals diffract the X-ray well and the diffraction intensity data were collected up to 1.6 A resolution. The crystal volume per unit mass, V(M) is 2.35 A3 Da(-1) with one molecule in the asymmetric unit, which corresponds to a solvent content of 47.7%.     

Crystallization and preliminary X-ray diffraction analysis of PD-L1, a highly glycosylated ribosome inactivating protein with DNase activity

PD-L1 is a highly glycosylated type 1 ribosome inactivating protein, from Phytolacca dioica leaves, with the peculiarity to act also as a DNase. PD-L1 has been successfully crystallized using vapour diffusion and seeding techniques. Crystals belong to the monoclinic C2 space group, with unit cell dimensions a=161.01, b=34.73, c=120.63 A, beta=127.99 degrees . Two molecules are present in the asymmetric unit. Phase determination has been achieved using molecular replacement.     

Crystallization and preliminary X-ray diffraction studies of dialkylglycine decarboxylase, a decarboxylating transaminase.

The pyridoxal phosphate-dependent enzyme dialkylglycine decarboxylase (E.C. 4.1.1.64) has been crystallized by vapor diffusion from a 15% polyethyleneglycol solution with sodium pyruvate as coprecipitant. The space group of the crystals is either P6(2)22 or the enantiomorph, P6(4)22, with one subunit of 46,500 Da per asymmetric unit. The unit cell has dimensions a = b = 152.7 A, c = 86.6 A, alpha = beta = 90 degrees, gamma = 120 degrees, and a solvent content of approximately 61%. diffraction extends to 2.3 A resolution.     

Crystallization and preliminary X-ray diffraction studies of the human erythrocyte bisphosphoglycerate mutase.

Bisphosphoglycerate mutase (EC 2.7.5.4) catalyzes the synthesis and breakdown of 2,3-diphosphoglycerate in red cells. The human enzyme, cloned and expressed in Escherichia coli has been crystallized in the rhombohedral space group R32 with a = b = c = 100.4 A and alpha = beta = gamma = 81.2 degrees. The asymmetric unit contains either a dimeric enzyme molecule, or a monomer.