Preliminary X-ray Diffraction Data Analysis Research Articles

Crystallization and preliminary X-ray crystallographic analysis of the sclerostin-neutralizing Fab AbD09097.

The secreted cystine-knot protein sclerostin was first identified from genetic screening of patients suffering from the rare bone-overgrowth diseases sclerosteosis and van Buchem disease. Sclerostin acts a negative regulator of bone growth through inhibiting the canonical Wnt signalling cascade by binding to and blocking the Wnt co-receptor LRP5/6. Its function in blocking osteoblastogenesis makes it an important target for osteoanabolic therapy approaches to treat osteoporosis, which is characterized by a progressive decrease in bone mass and density. In this work, the production, crystallization and preliminary X-ray diffraction data analysis of a sclerostin-neutralizing human Fab antibody fragment, AbD09097, obtained from a naive antibody library are reported. Crystals of the Fab AbD09097 belonged to space group P21, with unit-cell parameters a = 45.19, b = 78.49, c = 59.20 Å, β = 95.71° and diffracted X-rays to a resolution of 1.8 Å.

Crystallization and preliminary X-ray crystallographic analysis of a blue-light-absorbing proteorhodopsin

Proteorhodopsins (PRs), seven-transmembrane chromoproteins with retinal as a chromophore, are light-driven proton pumps. To elucidate the light-driven proton-pumping mechanism of PRs, a pET28a vector containing the blue-light-absorbing proteorhodopsin (BPR) gene was constructed and the protein was overexpressed in Escherichia coli. The protein was purified by immobilized metal-ion affinity chromatography (IMAC). The purified BPR D97N mutant protein (BPR_D97N) was crystallized using the vapour-diffusion method. Preliminary X-ray diffraction data analysis showed that the crystal belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 161.6, b = 168.6, c = 64.7 Å. A complete data set was collected to 3.3 Å resolution using synchrotron radiation on beamline X06 of the Swiss Light Source (SLS). Molecular replacement was unsuccessful. To solve the structure of BPR_D97N by experimental phasing, selenomethionine-substituted protein crystals were prepared. These crystals diffracted to 3.0 Å resolution and a complete data set was collected on beamline BL17U of the Shanghai Synchrotron Radiation Facility (SSRF). Heavy-atom substructure determination and phasing by SAD clearly showed that the crystal contained five molecules in the asymmetric unit, with a V(M) of 3.26 Å(3) Da(-1) and a solvent content of 62.3%.

Crystallization and preliminary X-ray analysis of CYP153C1 from Novosphingobium aromaticivorans DSM12444.

Cytochrome P450 (CYP) enzymes constitute a large family of haemoproteins that catalyze the monooxygenation of a great variety of endogenous and exogenous organic compounds. In common with other members of the CYP153 family of alkane hydroxylases, CYP153C1 from the oligotrophic bacterium Novosphingobium aromaticivorans DSM 12444 can bind linear alkanes such as heptane, octane and nonane. Here, the production, purification and crystallization of CYP153C1 and the collection of high-resolution diffraction data to 1.77 Å resolution are reported. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 61.0, b = 96.3, c = 149.8 Å, α = β = γ = 90.0°. Preliminary X-ray diffraction data analysis revealed that the asymmetric unit is most likely to contain two protein molecules.

Crystallization and preliminary crystallographic analysis of the central domain ofDrosophilaDribble, a protein that is essential for ribosome biogenesis. Addendum

Dribble (DBE) is a Drosophila protein that is essential for ribosome biogenesis. Bioinformatics analysis revealed a folded central domain of DBE which is flanked by structural disorder in the N- and C-terminal regions. The protein fragment spanning amino-acid residues 16-197 (DBE(16-197)) was produced for structural determination. In this report, the crystallization and preliminary X-ray diffraction data analysis of the DBE(16-197) protein domain are described. Crystals of DBE(16-197) were grown by the sitting-drop vapour-diffusion method at 289 K using ammonium phosphate as a precipitant. The crystals belonged to space group P2(1)2(1)2(1). Data were collected that extended to beyond 2 A resolution.

Expression, purification, crystallization and preliminary crystallographic analysis of PA3885 (TpbA) from Pseudomonas aeruginosa PAO1.

Biofilms are important in cell communication and growth in most bacteria and are also responsible for most human clinical infections and diseases. Quorum-sensing systems have been identified to be crucial for biofilm formation and regulation. PA3885 (TpbA), a tyrosine phosphatase, is reported to convert extracellular quorum-sensing signals into internal gene-cascade reactions that result in reduced biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. Here, PA3885 from P.aeruginosa PAO1 was expressed, purified and crystallized. Single crystals were studied by X-ray crystallography and native diffraction data were collected to 2.8 Å resolution. These crystals were determined to belong to space group C2. It was not possible to conclusively determine the number of proteins in the asymmetric unit from the preliminary X-ray diffraction data analysis alone and attempts to determine the crystal structure of PA3885 are currently under way.

Crystallization and preliminary X-ray diffraction data analysis of stenodactylin, a highly toxic type 2 ribosome-inactivating protein fromAdenia stenodactyla

Ribosome-inactivating proteins (RIPs) inhibit protein synthesis and induce cell death by removing a single adenine from a specific rRNA loop. They can be divided into two main groups: type 1 and type 2 RIPs. Type 1 RIPs are single-chain enzymes with N-glycosidase activity. Type 2 RIPs contain two chains (A and B) linked by a disulfide bond. The A chain has RIP enzymatic activity, whereas the B chain shows lectin activity and is able to bind to glycosylated receptors on the cell surface. Stenodactylin is a type 2 RIP from the caudex of Adenia stenodactyla from the Passifloraceae family that has been recently purified and characterized. It shows a strong enzymatic activity towards several substrates and is more cytotoxic than other toxins of the same type. Here, the crystallization and preliminary X-ray diffraction data analysis of stenodactylin are reported. This RIP forms crystals that diffract to high resolution (up to 2.15 A). The best data set was obtained by merging data collected from two crystals. Stenodactylin crystals belonged to the centred monoclinic space group C2 and contained two molecules in the asymmetric unit.

Purification, crystallization and preliminary X-ray analysis of cytochrome P450 219A1 fromNovosphingobium aromaticivoransDSM 12444

Cytochrome P450 enzymes catalyze a variety of reactions and are widely distributed in living organisms. In recent studies, the first members of five new families of cytochrome P450 enzymes have been identified, including cytochrome P450 219A1 (CYP219A1) from Novosphingobium aromaticivorans DSM 12444. It has also been reported that isolongifolen-9-one (C(15)H(22)O), a sesquiterpenoid ketone derivative, is a potential substrate for CYP219A1, inducing a >or=95% shift of the haem spin state to high spin upon binding. The CYP219A1 protein has been crystallized and single crystals have been studied by X-ray crystallography. Diffraction data were collected to 2.4 A resolution. The crystals belonged to space group P6, with unit-cell parameters a = 93.1, b = 93.1, c = 98.0 A. Preliminary X-ray diffraction data analysis revealed that the asymmetric unit contained one protein molecule.

Expression, purification and preliminary X-ray diffraction studies of VERNALIZATION1208–341fromArabidopsis thaliana

VERNALIZATION1 (VRN1) is required in the model plant Arabidopsis thaliana for the epigenetic suppression of the floral repressor FLC by prolonged cold treatment. Stable suppression of FLC accelerates flowering, a physiological process known as vernalization. VRN1 is a 341-residue DNA-binding protein that contains two plant-specific B3 domains (B3a and B3b), a putative nuclear localization sequence (NLS) and two putative PEST domains. VRN1(208-341) includes the second B3 domain and a region upstream that is highly conserved in the VRN1 orthologues of other dicotyledonous plants. VRN1(208-341) was crystallized by the hanging-drop method in 0.05 M sodium acetate pH 6.0 containing 1.0 M NaCl and 18%(w/v) PEG 3350. Preliminary X-ray diffraction data analysis revealed that the VRN1(208-341) crystal diffracted to 2.1 A and belonged to space group C2, with unit-cell parameters a = 105.2, b = 47.9, c = 61.2 A, alpha = 90.0, beta = 115.4, gamma = 90.0 degrees . Assuming that two molecules occupy the asymmetric unit, a Matthews coefficient of 2.05 A(3) Da(-1) and a solvent content of 40.1% were calculated.

Expression, purification and preliminary X-ray diffraction studies of the transcriptional factor PyrR from Bacillus halodurans.

The PyrR transcriptional regulator is widely distributed in bacteria. This RNA-binding protein is involved in the control of genes involved in pyrimidine biosynthesis, in which uridyl and guanyl nucleotides function as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of two crystal forms of Bacillus halodurans PyrR are reported. One of the forms belongs to the monoclinic space group P2(1) with unit-cell parameters a = 59.7, b = 87.4, c = 72.1 A, beta = 104.4 degrees , while the other form belongs to the orthorhombic space group P22(1)2(1) with unit-cell parameters a = 72.7, b = 95.9, c = 177.1 A. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of four and six monomers per asymmetric unit; a crystallographic tetramer is formed in both forms.

Purification, crystallization and preliminary X-ray crystallographic analysis of ST1022, a putative member of the Lrp/AsnC family of transcriptional regulators isolated fromSulfolobus tokodaiistrain 7

The Lrp/AsnC family of transcriptional regulators, also known as feast/famine transcriptional regulators, are widely distributed among bacteria and archaea. This family of proteins are likely to be involved in cellular metabolism, with exogenous amino acids functioning as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of ST1022, a member of the Lrp/AsnC family of proteins, is reported with and without exogenous glutamine as the effector molecule. The crystals of native ST1022 and of the putative complex belong to the tetragonal space group I422, with unit-cell parameters a = b = 103.771, c = 73.297 A and a = b = 103.846, c = 73.992 A, respectively. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of one monomer per asymmetric unit.

Crystallization and preliminary X-ray analysis of phage Mu activator protein C in a complex with promoter DNA

Bacteriophage Mu C protein is an activator of the four Mu late promoters that drive the expression of genes encoding DNA-modification as well as phage head and tail morphogenesis proteins. This report describes the purification and cocrystallization of wild-type and selenomethionine-substituted C protein with a synthetic late promoter P(sym), together with preliminary X-ray diffraction data analysis using SAD phasing. The selenomethionine peak data set was collected from a single crystal which diffracted to 3.1 A resolution and belonged to space group P4(1) or P4(3), with unit-cell parameters a = 68.9, c = 187.6 A and two complexes per asymmetric unit. The structure will reveal the amino acid-DNA interactions and any conformational changes associated with DNA binding.

Structural Insights into the Exchange Domain of Sec2p: Expression, Purification,Crystallization, and Preliminary X-Ray Diffraction Data Analysis

Sec2p is an essential yeast gene and is part of the cell polarization process that leads to budding. The N-terminal domain of sec2p (Sec2pN)--the guanine-nucleotide exchange factor for sec4p--has been expressed in Escherichia coli, purified, and crystallized. Crystals belong to the space group P2(1) with unit cell dimensions 178.1 x 98.4 x 180.0 A, beta = 91.7( degrees ), and diffract synchrotron-generated X-rays to better than 3.6 A resolution. Pseudo-precession plots reveal a Laue symmetry of 2/m, corresponding to the aforementioned space group, and unusual weak diffraction in the approximately 5-7 A resolution range. The Matthews number calculations for a typical crystal density suggest a range of 28 to 64 molecules per asymmetric unit. Self-rotation and native Patterson calculations demonstrate a pure helical array of protein subunits. Based on the X-ray diffraction data analysis and amino-acid sequence alignments, the paper presents a hypothetical model of the exchange domain of sec2p as a pair of coiled-coil helices that binds to sec4p and facilitates nucleotide disassociation.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of Rv2827c from Mycobacterium tuberculosis.

The hypothetical protein Rv2827c from Mycobacterium tuberculosis was cloned and heterologously expressed in Escherichia coli. It was purified using affinity and size-exclusion chromatographic techniques and then crystallized. Preliminary X-ray diffraction data analysis suggests the presence of two translationally related molecules in the asymmetric unit of the orthorhombic crystals.

Crystallization and preliminary crystallographic analysis of human common-type acylphosphatase.

Human acylphosphatase, an 11 kDa enzyme that catalyzes the hydrolysis of carboxyl phosphate bonds, has been studied extensively as a model system for amyloid-fibril formation. However, the structure is still not known of any isoform of human acylphosphatase. Here, the crystallization and preliminary X-ray diffraction data analysis of human common-type acylphosphatase are reported. Crystals of human common-type acylphosphatase have been grown by the sitting-drop vapour-diffusion method at 289 K using polyethylene glycol 4000 as precipitant. Diffraction data were collected to 1.45 A resolution at 100 K. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 42.58, b = 47.23, c = 57.26 A.