Trigonal Space Group P3 Research Articles

Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1.

The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl(2), 20% PEG 3350. Data were collected to 3.5 A resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 A. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease.

Fab crystallization and preliminary X-ray analysis of NC-1, an anti-HIV-1 antibody that recognizes the six-helix bundle core of gp41

NC-1 is a murine monoclonal antibody that specifically recognizes the six-helix bundle core of the human immunodeficiency virus type 1 (HIV-1) gp41. As such, it is a useful tool for probing gp41 conformations in HIV-1 membrane fusion. To establish the structural basis underlying the NC-1 specificity, X-ray crystallography was employed to solve its three-dimensional structure. To accomplish this, hybridoma-produced NC-1 antibody was first purified and digested with papain. Its Fab fragment was then purified using size-exclusion chromatography following Fc depletion using a Protein A affinity column. Finally, crystallization of NC-1 Fab was performed by the hanging-drop vapour-diffusion method and the protein was crystallized at pH 8.0 using PEG 6000 as precipitant. The results showed that the NC-1 Fab crystals belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 118.7, c = 106.0 A. There is one Fab molecule in the asymmetric unit, with 67.5% solvent content. An X-ray diffraction data set was collected at 3.2 A resolution and a clear molecular-replacement solution was obtained for solution of the structure.

Cesium Hydroperoxostannate: First Complete Structural Characterization of a Homoleptic Hydroperoxocomplex

The crystal structure of cesium hexahydroperoxostannate Cs(2)Sn(OOH)(6) is presented. The compound was characterized by single crystal and by powder X-ray diffraction, FTIR, (119)Sn MAS NMR, and TG-DTA. Cs(2)Sn(OOH)(6) crystallizes in the trigonal space group P3, a = 7.5575(4), c = 5.1050(6) A, V = 252.51(4) A(3), Z = 1, R(1) = 0.0120 (I > 2sigma(I)), wR(2) = 0.0293 (all data), and comprises cesium cations and slightly distorted octahedral [Sn(OOH)(6)](2-) anions lying on the threefold axis. The [Sn(OOH)(6)](2-) unit forms 12 interanion hydrogen bonds resulting in anionic chains spread along the c-axis. All six hydroperoxo ligands are crystallographically equivalent; O-O distances are 1.482(2), only slightly longer than the O-O distance in hydrogen peroxide. FTIR and (119)Sn MAS NMR reveal the similarity between all alkali hydroperoxostannates.

K 2Fe 2B 2O 7: A transparent nonlinear optical crystal with frustrated magnetism

A new noncentrosymmetric ferroborate crystal, K 2Fe 2B 2O 7, has been grown from high temperature melt. Structure solution from single crystal X-ray diffraction shows that the title compound crystallizes in a trigonal space group P321 with cell dimensions of a=8.7475(12) Å and c=8.5124(17) Å. In the structure, FeO 4 tetrahedron shares its three basal oxygen atoms with BO 3 triangles forming a two-dimensional layer in the ab plane and the layers are connected by the apical Fe–O bonds along the c direction. The crystal is transparent in the visible and near infrared region from 500 to 2000 nm with three pronounced absorption bands ascribed to d– d transitions of tetrahedrally coordinated Fe 3+ ions. Though, structurally analog to K 2Al 2B 2O 7, the further twisting of the BO 3 groups between adjacent layers reduces its optical nonlinearity to a second-harmonic generation intensity of about 0.4 times that of KDP. Spin-glass behavior is observed at 20 K which is probably due to geometrically magnetic frustration of the triangular Fe net in the ab plane.

Overexpression, crystallization and preliminary X-ray crystallographic analysis ofD-ribose-5-phosphate isomerase fromClostridium thermocellum

Rare sugars are used for many industrial and medical purposes and are produced by the interconversion between aldoses and ketoses catalyzed by sugar and sugar-phosphate isomerases. Recently, Clostridium thermocellum d-ribose-5-phosphate isomerase (CTRPI), an aldose-ketose isomerase, was cloned in order to synthesize d-allose and its substrate specificity was further characterized for industrial usage. CTRPI has a novel substrate specificity that differs from those of other isomerases, which have broad substrate specificities. CTRPI prefers aldose substrates such as l-talose, d-ribose and d-allose. CTRPI was purified and crystallized in order to determine its three-dimensional structure and thus to elucidate its enzymatic reaction mechanism and understand its substrate specificity. The crystal belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 69.5, c = 154.4 angstrom, and diffracted to 1.9 angstrom resolution. According to Matthews coefficient calculations, the crystallographic structure consists of a dimer in the asymmetric unit, with a V(M) of 3.2 angstrom(3) Da(-1) and a solvent content of 61.7%.

Purification, crystallization and preliminary X-ray analysis of FliT, a bacterial flagellar substrate-specific export chaperone

The assembly process of the bacterial flagellum is coupled to flagellar gene expression. FliT acts not only as a flagellar type III substrate-specific export chaperone for the filament-capping protein FliD but also as a negative regulator that suppresses flagellar gene expression through its specific interaction with the master regulator FlhD(4)C(2) complex. In this study, FliT of Salmonella enterica serovar Typhimurium was expressed, purified and crystallized. Crystals of SeMet FliT were obtained by the sitting-drop vapour-diffusion technique with potassium/sodium tartrate as the precipitant. The crystals grew in the trigonal space group P3(1)21 or P3(2)21 and diffracted to 3.2 A resolution. The anomalous difference Patterson map of the SeMet FliT crystal showed significant peaks in its Harker sections, indicating the usefulness of the derivative data for structure determination.

Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 9.

Adeno-associated virus (AAV) serotype 9, which is under development for gene-delivery applications, shows significantly enhanced capsid-associated transduction efficiency in muscle compared with other AAV serotypes. With the aim of characterizing the structural determinants of this property, the purification, crystallization and preliminary X-ray crystallographic analyses of the AAV9 viral capsid are reported. The crystals diffracted X-rays to 2.8 A resolution using synchrotron radiation and belonged to the trigonal space group P3(2), with unit-cell parameters a = b = 251.0, c = 640.0 A. There are three complete viral capsids in the crystal unit cell. The orientation and position of the asymmetric unit capsid have been determined by molecular-replacement methods and structure determination is in progress.

Purification, crystallization and preliminary X-ray analysis of the beta-lactamase Oih-1 from Oceanobacillus iheyensis.

Bacterial resistance to the beta-lactam family of antibiotics is primarily the result of the deactivation of the drugs by beta-lactamase enzymes. The gene encoding the proficient beta-lactamase Oih-1 from the alkaliphilic and halotolerant Gram-positive bacterium Oceanobacillus iheyensis has been cloned and the mature wild-type protein (comprising 274 amino-acid residues) has been expressed in Escherichia coli and subsequently purified to homogeneity. Oih-1 crystallized in two crystal forms both belonging to the trigonal space group P3(1)21 but with distinctly different unit-cell parameters. Synchrotron diffraction data were collected to high resolution (1.65-1.75 A) from both crystal forms on beamlines BL7-1 and BL11-1 at SSRL (Stanford, California, USA).

Crystallization and preliminary X-ray analysis of carnein, a serine protease from Ipomoea carnea.

Carnein is an 80 kDa subtilisin-like serine protease from the latex of the plant Ipomoea carnea which displays an exceptional resistance to chemical and thermal denaturation. In order to obtain the first crystal structure of a plant subtilisin and to gain insight into the structural determinants underlying its remarkable stability, carnein was isolated from I. carnea latex, purified and crystallized by the hanging-drop vapour-diffusion method. A data set was collected to 2.0 A resolution in-house from a single crystal at 110 K. The crystals belonged to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 126.9, c = 84.6 A, alpha = beta = 90, gamma = 120 degrees. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient is 2.46 A(3) Da(-1), corresponding to a solvent content of 50%. Structure determination of the enzyme is in progress.

Determining the DUF55-domain structure of human thymocyte nuclear protein 1 from crystals partially twinned by tetartohedry

Human thymocyte nuclear protein 1 contains a unique DUF55 domain consisting of 167 residues (55-221), but its cellular function remains unclear. Crystals of DUF55 belonged to the trigonal space group P3(1), but twinning caused the data to approach apparent 622 symmetry. Two data sets were collected to 2.3 A resolution. Statistical analysis confirmed that both data sets were partially twinned by tetartohedry. Tetartohedral twin fractions were estimated. After the structure had been determined, only one twofold axis of rotational pseudosymmetry was found in the crystal structure. Using the DALI program, a YTH domain, which is a potential RNA-binding domain from human YTH-domain-containing protein 2, was identified as having the most similar three-dimensional fold to that of DUF55. It is thus implied that DUF55 might be a potential RNA-related domain.

Expression, purification, crystallization and preliminary crystallographic study of the SRA domain of the human UHRF1 protein.

Human UHRF1 belongs to the unique mammalian family of proteins which contain a SET- and RING finger-associated (SRA) domain. This 180-residue domain has been reported to play key roles in the functions of the protein. It allows UHRF1 to bind methylated DNA, histone deacetylase 1 and DNA methyltransferase 1, suggesting a bridge between DNA methylation and the histone code. No structural data is available for any SRA domain. Native and SeMet-labelled SRA domains of human UHRF1 were overexpressed in Escherichia coli cells, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. A complete MAD data set was collected to 2.2 A resolution at 100 K. Crystals of the SeMet-labelled protein belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 53.78, c = 162.05 A.

Purification, crystallization and preliminary X-ray diffraction analysis of a cystathionine β-synthase domain-containing protein, CDCP2, fromArabidopsis thaliana

Cystathione beta-synthase domain-containing protein 2 (CDCP2) from Arabidopsis thaliana has been overexpressed and purified to homogeneity. As an initial step towards three-dimensional structure determination, crystals of recombinant CDCP2 protein have been obtained using polyethylene glycol 8000 as a precipitant. The crystals diffracted to 2.4 A resolution using synchrotron radiation and belonged to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 56.360, c = 82.596 A, alpha = beta = 90, gamma = 120 degrees . The asymmetric unit contains one CDCP2 molecule and the solvent content is approximately 41%.

Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-beta-D-xylanase 10B in complex with xylohexaose.

The cellulosome of Clostridium thermocellum is a highly organized multi-enzyme complex of cellulases and hemicellulases involved in the hydrolysis of plant cell-wall polysaccharides. The bifunctional multi-modular xylanase Xyn10B is one of the hemicellulase components of the C. thermocellum cellulosome. The enzyme contains an internal glycoside hydrolase family 10 catalytic domain (GH10) and a C-terminal family 1 carbohydrate esterase domain (CE1). The N-terminal moiety of Xyn10B (residues 32-551), comprising a carbohydrate-binding module (CBM22-1) and the GH10 E337A mutant, was crystallized in complex with xylohexaose. The crystals belong to the trigonal space group P3(2)21 and contain a dimer in the asymmetric unit. The crystals diffracted to beyond 2.0 A resolution.

A Preliminary X-Ray Study of a Refolded PTS EIIBfruc Protein from Escherichia coli

The phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) catalyzes the phosphorylation and transportation of its sugar substrates. A sugar-specific enzyme II complex involved in the PTS finally functions to translocate substrates across the membrane. A PTS EIIB(fruc) protein, a fructose specific EIIB subunit, from Escherichia coli has been cloned, expressed, refolded, purified, and crystallized. The synchrotron data were collected to 2.6 A from the crystal of a selenomethionine substitute PTS EIIB(fruc) protein. The crystal belongs to the primitive trigonal space group P3(1)21, with unit-cell parameters of a = 33.4 A, b = 33.4 A, c = 154.0 A, and beta = 120.0 degrees . A full structure determination is under way to provide insights into the structure-function relationships of this protein.

Crystallization and preliminary X-ray crystallographic studies of human voltage-dependent anion channel isoform I (HVDAC1)

The major channel by which metabolites can pass through the outer mitochondrial membrane is formed by the voltage-dependent anion-channel (VDAC) family. Functionally, VDAC is involved in the limited exchange of ATP, ADP and small hydrophilic molecules across the outer membrane. Moreover, there is compelling evidence that VDAC isoforms in mammals may act in the cross-talk between mitochondria and the cytoplasm by direct interaction with enzymes involved in energy metabolism and proteins involved in mitochondrial-induced apoptosis. To obtain a high-resolution structure of this channel, human VDAC protein isoform I was overproduced in Escherichia coli. After refolding and testing the correct fold using circular dichroism, a subsequent broad-range screening in different detergents resulted in a variety of crystals which diffracted to 3.5 A resolution. The crystal lattice belongs to the trigonal space group P321, with unit-cell parameters a = 78.9, c = 165.7 A and one monomer in the asymmetric unit.

Synthesis, Structure, and Magnetic Properties of [(S)-[PhCH(CH3)N(CH3)3]][Mn(CH3CN)2/3Cr(ox)3]·(CH3CN)_(solvate), a 2D Chiral Magnet Containing a Quaternary Ammonium Chiral Cation

The synthesis, structure, and magnetic properties of a novel oxalate-based bimetallic magnet obtained by using the chiral (S)-trimethyl-(1-phenyl-ethyl)-ammonium, ((S)-[PhCH(CH3)N(CH3)3](+)), cation as template is reported. This compound can be formulated as [(S)-[PhCH(CH3)N(CH3)3]][Mn(CH3CN)2/3Cr(ox)3] x (CH3CN)_(solvate), and it crystallizes in the chiral trigonal space group P3. It shows a distorted two-dimensional honeycomb structure formed by Mn(II) and Cr(III) ions connected through oxalate anions with [(S)-[PhCH(CH3)N(CH3)3](+) cations and solvent molecules intercalated between the oxalate layers. Two-thirds of the Mn(II) ions of the honeycomb anionic network are heptacoordinated. This compound behaves as a soft ferromagnet with an ordering temperature of 5.6 K.

Crystal structure of BaCa 2MgSi 2O 8 and the photoluminescent properties activated by Eu 2+

Rietveld refinements of X-ray powder diffraction data have confirmed the crystal structure of BaCa 2MgSi 2O 8 prepared by a standard solid-state method. The final reliable factors were R wp=10.91%, R p=8.10%, R I=2.71%, and R F=1.14%. BaCa 2MgSi 2O 8 crystallizes in the trigonal space group P3¯m1 (no. 164) with a=5.430(3) Å and c=6.807(2) Å. The oxide has a layered structure constructed from the unit layers built up by corner-sharing MgO 6 octahedra and SiO 4 tetrahedra. Ba and Ca atoms occupy the distinct crystallographic sites; Ba atom is sited in the interlayer space and Ca atom is embedded in the layer framework. This structure was not disrupted by doping of Eu 2+ ions. The Eu 2+-doped BaCa 2MgSi 2O 8 exhibited an intense blue emission based on 5d–4f electron transitions of Eu 2+ ions under 254 nm excitation. This emission has a sufficient chromaticity as a blue phosphor. An additional analysis of the emission spectra using an empirical formula indicates that Eu 2+ is distributed into both Ba and Ca sites.

Helical Polymer 1/∞[P2Se62-]: Strong Second Harmonic Generation Response and Phase-Change Properties of Its K and Rb Salts

The selenophosphates A2P2Se6 (A = K, Rb) crystallize in the chiral trigonal space group P3121, with a = 7.2728(9) A, c = 18.872(4) A, and Z = 3 at 298(2) K and a = 14.4916(7) A, c = 18.7999(17) A, and Z = 12 at 173(2) K for K+ salt and a = 7.2982(5) A, c = 19.0019(16) A, and Z = 3 at 100(2) K for Rb+ salt. The A2P2Se6 feature parallel one-dimensional helical chains of 1/infinity[P2Se62-] which depict an oxidative polymerization of the ethane-like [P2Se6]4- anion. On cooling well below room temperature K2P2Se6 exhibits a displacive phase transition to a crystallographic subgroup and forms a superstructure with a cell doubling along the a- and b-axes. The Rb analogue does not exhibit the phase transition. The compounds are air stable and show reversible glass-crystal phase-change behavior with a band gap red shift of 0.11 and 0.22 eV for K+ and Rb+ salts, respectively. Raman spectroscopy, 31P magic angle spinning solid-state NMR, and pair distribution function (PDF) analysis for crystalline and glassy K2P2Se6 give further understanding of the phase transition and the local structure of the amorphous state. K2P2Se6 exhibits excellent mid-IR transparency and a strong second harmonic generation (SHG) response. The SHG response is type-I phase-matchable and in the wavelength range of 1000-2000 nm was measured to be 50 times larger than that of the commercially used material AgGaSe2. Glassy K2P2Se6 also exhibits an SHG response without the application of electric field poling. In connection with the NLO properties the thermal expansion coefficients for K2P2Se6 are reported.

Purification, crystallization and preliminary X-ray diffraction analysis of royal palm tree (Roystonea regia) peroxidase.

Royal palm tree peroxidase (RPTP), which was isolated from Roystonea regia leaves, has an unusually high stability that makes it a promising candidate for diverse applications in industry and analytical chemistry [Caramyshev et al. (2005), Biomacromolecules, 6, 1360-1366]. Here, the purification and crystallization of this plant peroxidase and its X-ray diffraction data collection are described. RPTP crystals were obtained by the hanging-drop vapour-diffusion method and diffraction data were collected to a resolution of 2.8 A. The crystals belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 116.83, c = 92.24 A, and contain one protein molecule per asymmetric unit. The V(M) value and solvent content are 4.07 A3 Da(-1) and 69.8%, respectively.

Expression, purification, crystallization and preliminary X-ray analysis of the ligand-binding domain of metabotropic glutamate receptor 7.

Glutamate is the major excitatory neurotransmitter and its metabotropic glutamate receptor (mGluR) plays an important role in the central nervous system. The ligand-binding domain (LBD) of mGluR subtype 7 (mGluR7) was produced using the baculovirus expression system and purified from the culture medium. The purified protein was characterized by gel-filtration chromatography, SDS-PAGE and a ligand-binding assay. Crystals of mGluR7 LBD were grown at 293 K by the hanging-drop vapour-diffusion method. The crystals diffracted X-rays to 3.30 A resolution using synchrotron radiation and belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 92.4, c = 114.3 A. Assuming the presence of one protomer per crystallographic asymmetric unit, the Matthews coefficient V(M) was calculated to be 2.5 A3 Da(-1) and the solvent content was 51%.