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1.  Crystallization and preliminary crystallographic analysis of 2-aminophenol 1,6-dioxygenase complexed with substrate and with an inhibitor 
The crystallization of 2-aminophenol 1,6-dioxygenase in complexes with its substrate and with an inhibitor is reported.
Dioxygen activation implemented by nonhaem FeII enzymes containing the 2-­His-1-carboxylate facial triad has been extensively studied in recent years. Extradiol dioxygenase is the archetypal member of this superfamily and catalyzes the oxygenolytic ring opening of catechol analogues. Here, the crystallization and preliminary X-ray analysis of 2-aminophenol 1,6-dioxygenase, an enzyme representing a minor subset of extradiol dioxygenases that catalyze the fission of 2-aminophenol rather than catecholic compounds, is reported. Crystals of the holoenzyme with FeII and of complexes with the substrate 2-aminophenol and the suicide inhibitor 4-nitrocatechol were grown using the cocrystallization method under the same conditions as used for the crystallization of the apoenzyme. The crystals belonged to space group C2 and diffracted to 2.3–2.7 Å resolution; the crystal that diffracted to the highest resolution had unit-cell parameters a = 270.24, b = 48.39, c = 108.55 Å, β = 109.57°. All X-ray data sets collected from diffraction-quality crystals were suitable for structure determination.
doi:10.1107/S1744309112038705
PMCID: PMC3515376  PMID: 23143244
2-aminophenol 1,6-dioxygenase; extradiol dioxygenases; 2-aminophenol; catechol
2.  Crystallization and preliminary crystallographic studies of a cysteine protease inhibitor from the human nematode parasite Ascaris lumbricoides  
A recombinant cysteine protease inhibitor from the human nematode parasite A. lumbricoides has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.1 Å resolution.
The cysteine protease inhibitor from Ascaris lumbricoides, a roundworm that lives in the human intestine, may be involved in the suppression of human immune responses. Here, the molecular cloning, protein expression and purification, preliminary crystallization and crystallographic characterization of the cysteine protease inhibitor from A. lumbricoides are reported. The rod-shaped crystal belonged to space group C2, with unit-cell parameters a = 99.40, b = 37.52, c = 62.92 Å, β = 118.26°. The crystal diffracted to 2.1 Å resolution and contained two molecules in the asymmetric unit.
doi:10.1107/S1744309110050773
PMCID: PMC3034614  PMID: 21301092
cysteine protease inhibitors; nematode parasites; Ascaris lumbricoides
3.  Crystallization and preliminary crystallographic analysis of Gibberella zeae extracellular lipase 
G. zeae extracellular lipase has been overexpressed, purified and crystallized. Diffraction data were collected to 2.8 Å resolution.
Fusarium head blight, one of the most destructive crop diseases, is mainly caused by Fusarium graminearum (known in its sexual stage as Gibberella zeae). F. graminearum secretes various extracellular enzymes that have been hypothesized to be involved in host infection. One of the extracellular enzymes secreted by this organism is the G. zeae extracellular lipase (GZEL), which is encoded by the FGL1 gene. In order to solve the crystal structure of GZEL and to gain a better understanding of the biological functions of the protein and of possible inhibitory mechanisms of lipase inhibitors, recombinant GZEL was crystallized at 291 K using PEG 3350 as a precipitant. A data set was collected to 2.8 Å resolution from a single flash-cooled crystal (100 K). The crystal belonged to space group P212121, with unit-cell parameters a = 78.4, b = 91.0, c = 195.8 Å, α = β = γ = 90°. The presence of four molecules was assumed per asymmetric unit, which gave a Matthews coefficient of 2.6 Å3 Da−1.
doi:10.1107/S1744309108019283
PMCID: PMC2531274  PMID: 18765911
extracellular lipases; Fusarium graminearum; Gibberella zeae; fusarium head blight
4.  Preliminary crystallographic analysis of glyceraldehyde-3-phosphate dehydrogenase 3 from Saccharomyces cerevisiae  
The crystallization and preliminary X-ray diffraction analysis of glyceraldehyde-3-phosphate dehydrogenase 3 from S. cerevisiae is reported.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an important enzyme in the glycolytic pathway. In addition to its conventional metabolic role, GAPDH has been identified to possess diverse cellular functions. In this study, glyceraldehyde-3-phosphate dehydrogenase 3, the third isoform of GAPDH from Saccharomyces cerevisiae, was cloned, expressed, purified and crystallized. The crystals belonged to space group I4122, with unit-cell parameters a = b = 116.13, c = 119.21 Å. X-ray diffraction data were collected to a resolution of 2.6 Å. The structure was solved by molecular replacement and refinement is in progress.
doi:10.1107/S1744309112028989
PMCID: PMC3412788  PMID: 22869137
glyceraldehyde-3-phosphate dehydrogenases; Saccharomyces cerevisiae
5.  Purification, crystallization and preliminary X-ray diffraction analysis of the effector protein PevD1 from Verticillium dahliae  
The overexpression, purification, crystallization and preliminary X-ray diffraction analysis of protein elicitor PevD1 from Verticillium dahliae are reported.
The effector protein PevD1 from the pathogenic fungus Verticillium dahliae was purified and crystallized using the hanging-drop vapour-diffusion method. Native crystals appeared in a solution consisting of 4.0 M sodium formate. A native data set was collected at 1.9 Å resolution at 100 K using an in-house X-ray source. Because of the absence of useful methinione in the protein sequence, derivative crystals that contained iodine were obtained by soaking in 1.25 M potassium iodide, and a data set that contained anomalous signal was collected using the same X-ray facility at a wavelength of 1.54 Å. The single-wavelength anomalous dispersion method was used to successfully solve the structure based on the anomalous signal generated from iodine.
doi:10.1107/S1744309112020556
PMCID: PMC3388926  PMID: 22750869
PevD1; effector proteins; Verticillium dahliae
6.  Preliminary crystallographic analysis of the RNA-binding domain of HuR and its poly(U)-binding properties 
Here, the recombinant ARE-binding region of HuR (residues 18–186) was crystallized in space group P21212, with unit-cell parameters a = 41.2, b = 133.1, c = 31.4 Å.
Human antigen R (HuR), a ubiquitously expressed member of the Hu protein family, is an important post-transcriptional regulator which has three RNA-recognition motif (RRM) domains. The two tandem N-terminal RRM domains can selectively bind to the AU-rich element (ARE), while the third one interacts with the poly(A) tail and other proteins. Here, the recombinant ARE-binding region of HuR (residues 18–186) was crystallized in space group P21212, with unit-cell parameters a = 41.2, b = 133.1, c = 31.4 Å. X-ray diffraction data were collected to a resolution of 2.8 Å. Mutagenesis analysis and SPR assays revealed its poly(U)-binding properties.
doi:10.1107/S1744309110052930
PMCID: PMC3087637  PMID: 21543858
human antigen R; RNA-recognition motif domains; poly(U) binding; post-transcriptional regulation
7.  Structure of the putative dihydroorotate dehydrogenase from Streptococcus mutans  
The crystal structure of SMU.595, a putative dihydroorotate dehydrogenase (DHOD) from S. mutans, is reported at 2.4 Å resolution.
Streptococcus mutans is one of the pathogenic species involved in dental caries, especially in the initiation and development stages. Here, the crystal structure of SMU.595, a putative dihydroorotate dehydrogenase (DHOD) from S. mutans, is reported at 2.4 Å resolution. DHOD is a flavin mononucleotide-containing enzyme which catalyzes the oxidation of l-dihydroorotate to orotate, which is the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. The reductive lysine-methylation procedure was applied in order to improve the diffraction qualities of the crystals. Analysis of the S. mutans DHOD crystal structure shows that this enzyme is a class 1A DHOD and also suggests potential sites that could be exploited for the design of highly specific inhibitors using the structure-based chemotherapeutic design technique.
doi:10.1107/S1744309110048414
PMCID: PMC3034605  PMID: 21301083
dihydroorotate dehydrogenases; Streptococcus mutans; pyrimidine biosynthesis
8.  Expression, crystallization and preliminary X-ray analysis of the phosphoribosylglycinamide formyltransferase from Streptococcus mutans  
Phosphoribosylglycinamide formyltransferase (PurN) from Streptococcus mutans was expressed in E. coli, purified and studied crystallographically.
Phosphoribosylglycinamide formyltransferase (PurN) from Streptococcus mutans was recombinantly expressed in Escherichia coli. An effective purification protocol was established. The purified protein, which had a purity of >95%, was identified by SDS–PAGE and MALDI–TOF MS. The protein was crystallized using the vapour-diffusion method in hanging-drop mode with PEG 3350 as the primary precipitant. X-ray diffraction data were collected to 2.1 Å resolution. Preliminary X-ray analysis indicated that the crystal belonged to space group P212121, with unit-cell parameters a = 52.25, b = 63.29, c = 131.81 Å.
doi:10.1107/S1744309110053170
PMCID: PMC3034630  PMID: 21301108
Streptococcus mutans; PurN; phosphoribosylglycinamide formyltransferases
9.  Preliminary X-ray crystallographic analysis of the glycosyltransferase from a marine Streptomyces species 
The recombinant glycosyltransferase ElaGT from the elaiophylin-producing marine Streptomyces sp. SCSIO 01934 has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.9 Å resolution.
ElaGT is a glycosyltransferase from a marine Streptomyces species that is involved in the biosynthesis of elaiophylin. Here, the molecular cloning, protein expression and purification, preliminary crystallization and crystallographic characterization of ElaGT are reported. The rod-shaped crystals belonged to space group P2122, with unit-cell parameters a = 66.7, b = 131.7, c = 224.6 Å, α = 90, β = 90, γ = 90°. Data were collected to 2.9 Å resolution. A preliminary molecular-replacement solution implied the presence of two ElaGT molecules in the asymmetric unit.
doi:10.1107/S1744309110048268
PMCID: PMC3079993  PMID: 21206045
ElaGT; glycosyltransferases; Streptomyces sp. SCSIO 01934
10.  Structure of orotate phosphoribosyltransferase from the caries pathogen Streptococcus mutans  
The crystal structure of OPRTase from the caries pathogen Streptococcus mutans is reported at 2.4 Å resolution.
Orotate phosphoribosyltransferase (OPRTase) catalyzes the OMP-forming step in de novo pyrimidine-nucleotide biosynthesis. Here, the crystal structure of OPRTase from the caries pathogen Streptococcus mutans is reported at 2.4 Å resolution. S. mutans OPRTase forms a symmetric dimer and each monomer binds two sulfates at the active sites. The structural symmetry of the sulfate-binding sites and the missing loops in this structure are consistent with a symmetric catalysis mechanism.
doi:10.1107/S1744309110009243
PMCID: PMC2864676  PMID: 20445243
orotate phosphoribosyltransferase; Streptococcus mutans
11.  Structure of Bacillus amyloliquefaciens α-amylase at high resolution: implications for thermal stability 
The crystal structure of B. amyloliquefaciens α-amylase (BAA) at 1.4 Å resolution revealed ambiguities in the thermal adaptation of homologous proteins in this family.
The crystal structure of Bacillus amyloliquefaciens α-amylase (BAA) at 1.4 Å resolution revealed ambiguities in the thermal adaptation of homologous proteins in this family. The final model of BAA is composed of two molecules in a back-to-back orientation, which is likely to be a consequence of crystal packing. Despite a high degree of identity, comparison of the structure of BAA with those of other liquefying-type α-amylases indicated moderate discrepancies at the secondary-structural level. Moreover, a domain-displacement survey using anisotropic B-factor and domain-motion analyses implied a significant con­tribution of domain B to the total flexibility of BAA, while visual inspection of the structure superimposed with that of B. licheniformis α-amylase (BLA) indicated higher flexibility of the latter in the central domain A. Therefore, it is suggested that domain B may play an important role in liquefying α-­amylases, as its rigidity offers a substantial improvement in thermostability in BLA compared with BAA.
doi:10.1107/S1744309109051938
PMCID: PMC2815676  PMID: 20124706
α-amylases; thermostability; flexibility; alignment
12.  Crystallization and preliminary crystallographic studies of the single-chain variable fragment of antibody chA21 in complex with an N-terminal fragment of ErbB2 
An antibody–antigen complex consisting of a single-chain variable fragment of the potential therapeutic antibody chA21 and an N-terminal fragment (residues 1–192) of the human ErbB2 extracellular domain was expressed, purified and crystallized. X-ray diffraction data were collected to 2.45 Å resolution.
ErbB2 is a transmembrane tyrosine kinase, the overexpression of which causes abnormality and disorder in cell signalling and leads to cell transformation. Previously, an anti-ErbB2 single-chain chimeric antibody chA21 that specifically inhibits the growth of ErbB2-overexpressing cancer cells in vitro and in vivo was developed. Here, an antibody–antigen complex consisting of the single-chain variable fragment (scFv) of chA21 and an N-terminal fragment (residues 1–192, named EP I) of the ErbB2 extracellular domain was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.45 Å resolution from a single flash-cooled crystal; the crystal belonged to space group P212121.
doi:10.1107/S1744309109020107
PMCID: PMC2705636  PMID: 19574641
chA21; ErbB2; antibodies; antigens
13.  Preliminary X-ray crystallographic studies of Bacillus subtilis SpeA protein 
In order to further illustrate the catalytic mechanism of arginine decarboxylase by determining the three-dimensional structure of the enzyme the speA gene was amplified from B. subtilis genomic DNA and cloned. The enzyme was expressed in Escherichia coli and purified to homogeneity by nickel-chelation chromatography followed by size-exclusion chromatography. High-quality crystals were obtained using the hanging-drop vapour-diffusion method at 298 K.
The speA gene in Bacillus subtilis encodes arginine decarboxylase, which catalyzes the conversion of arginine to agmatine. Arginine decarboxylase is an important enzyme in polyamine metabolism in B. subtilis. In order to further illustrate the catalytic mechanism of arginine decarboxylase by determining the three-dimensional structure of the enzyme, the speA gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET-28a(+). SpeA was expressed in Escherichia coli and purified to homogeneity by nickel-chelation chromatography followed by size-exclusion chromatography. High-quality crystals were obtained using the hanging-drop vapour-diffusion method at 289 K. The best crystal diffracted to 2.0 Å resolution and belonged to space group P21, with unit-cell parameters a = 86.4, b = 63.3 c = 103.3 Å, β = 113.9°.
doi:10.1107/S1744309109003856
PMCID: PMC2650450  PMID: 19255484
SpeA; Bacillus subtilis; arginine decarboxylases
14.  Crystallization and preliminary crystallographic studies of the W2 domain of Drosophila melanogaster eukaryotic translation initiation factor 5C domain-containing protein 
The crystallization and preliminary crystallographic studies of the carboxy-terminal domain of D. melanogaster eukaryotic translation initiation factor 5C domain-containing protein are reported.
The Drosophila melanogaster eukaryotic translation initiation factor 5C domain-containing protein (ECP) is composed of two independently folded domains which belong to the basic leucine-zipper and W2 domain-containing protein (BZW) family. Based on the sequence similarity between the C-terminal W2 domain of ECP and some eukaryotic translation initiation factors (such as eIF2B∊, eIF4γ, eIF5 etc.), ECP has been speculated to participate in the translation initiation process. Structural information on the C-terminal W2 domain of ECP would be helpful in understanding the specific cellular function of this protein. Here, the W2 domain of ECP was expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 2.70 Å resolution and belonged to space group I4, with unit-cell parameters a = b = 81.05, c = 57.44 Å. The Matthews coefficient suggested that there was one molecule per asymmetric unit in the crystal.
doi:10.1107/S1744309112036512
PMCID: PMC3515371  PMID: 23143239
ECP; eIF5C; W2 domain; Drosophila melanogaster
15.  Crystallization and preliminary X-ray diffraction analysis of alanine racemase from Pseudomonas putida YZ-26 
A recombinant alanine racemase from the Pseudomonas putida YZ-26, has been crystallized by the sitting-drop vapor-diffusion method and X-ray diffraction data were collected to 2.4 Å.
A recombinant form of alanine racemase (Alr) from Pseudomonas putida YZ-26 has been crystallized by the sitting-drop vapour diffusion method. X-ray diffraction data were collected to 2.4 Å resolution. The crystals belong to the space group C2221, with unit-cell parameters a = 118.08, b = 141.86, c = 113.83 Å, and contain an Alr dimer in the asymmetric unit. The Matthews coefficient and the solvent content were calculated to be 2.8 Å3 Da−1 and approximately 50%, respectively.
doi:10.1107/S1744309112036974
PMCID: PMC3497987  PMID: 23027757
alanine racemase; Pseudomonas putida YZ-26; d-alanine
16.  Structure determination by multiple-wavelength anomalous dispersion (MAD) at the Pr L III edge 
A successful MAD experiment has been conducted at the Pr L III edge on HZB beamline BL14.2.
The use of longer X-ray wavelengths in macromolecular crystallography has grown significantly over the past few years. The main reason for this increased use of longer wavelengths has been to utilize the anomalous signal from sulfur, providing a means for the experimental phasing of native proteins. Here, another possible application of longer X-ray wavelengths is presented: MAD at the L III edges of various lanthanide compounds. A first experiment at the L III edge of Pr was conducted on HZB MX beamline BL14.2 and resulted in the successful structure determination of the C-terminal domain of a spliceosomal protein. This experiment demonstrates that L III edges of lanthanides constitute potentially attractive targets for long-wavelength MAD experiments.
doi:10.1107/S1744309112025456
PMCID: PMC3412789  PMID: 22869138
long-wavelength MAD; MAD phasing; lanthanide ions
17.  Crystallization and preliminary X-ray studies of ferric uptake regulator from Magnetospirillum gryphiswaldense  
Crystals of apo and SeMet ferric uptake regulator from the magnetotactic bacterium M. gryphiswaldense MSR-1 were obtained and optimized to obtain high-quality diffraction data. Diffraction data sets were collected and processed at 1.58 and 1.9 Å resolution, respectively.
Magnetosomes in magnetotactic bacteria have been widely used in studies of magnetic domains and in commercial applications. The iron content of magnetotactic bacteria is ∼100 times higher than that of Escherichia coli. Magnetospirillum gryphiswaldense MSR-1 can still take up iron even at high intracellular concentrations. Ferric uptake regulator (Fur) is a global iron-responsive regulator that affects magnetosome formation, iron transport and oxygen metabolism. However, the mechanism of iron uptake and homeostasis by M. gryphiswaldense MSR-1 Fur is not clear. Here, the expression, purification and crystallization of apo and SeMet Fur from M. gryphiswaldense MSR-1 are reported. The crystals belonged to space group C2. Matthews coefficient analysis and size-exclusion chromatography showed that the asymmetric unit probably contains one dimer of Fur. Diffraction data were optimized to 1.58 Å resolution for apo Fur and to 1.9 Å resolution for SeMet Fur.
doi:10.1107/S1744309112024311
PMCID: PMC3412769  PMID: 22869118
ferric uptake regulator; Magnetospirillum gryphiswaldense MSR-1; magnetotactic bacteria
18.  Crystallization and preliminary X-ray diffraction analysis of a novel GH120 β-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-YS485 
Xylosidase C from Thermoanaerobacterium saccharolyticum, the first enzyme classified as GH120 family, has been successfully crystallized. The crystal diffracted to a resolution of 2.2 Å.
Xylosidases hydrolyze xylopolymers at the nonreducing end to free xylose units. The β-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-­YS485 was expressed in Escherichia coli and the recombinant protein was purified and crystallized. A BLASTP search with the XylC protein sequence showed that no similar structure had previously been solved. XylC was classified as a member of the new glycoside hydrolase family GH120 according to the CAZy website (http://www.cazy.org/). Crystals belonging to the monoclinic space group P21, with unit-cell parameters a = 88.36, b = 202.20, c = 99.87 Å, β = 99.04°, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.2 Å resolution. Structure determination using MIR and MAD methods is in progress.
doi:10.1107/S1744309112025328
PMCID: PMC3412772  PMID: 22869121
thermophilic enzymes; xylan; β-xylosidases
19.  Expression, crystallization and preliminary crystallographic data analysis of PigF, an O-­methyltransferase from the prodigiosin-synthetic pathway in Serratia  
The last step in the biosynthetic pathway of 4-methoxy-2,2′-bipyrrole-5-­carbaldehyde (MBC) is catalyzed by PigF, which transfers a methyl group to 4-­hydroxy-2,2′-bipyrrole-5-carbaldehyde (HBC) to form the terminal product MBC, but its catalytic mechanism is not known. To elucidate its mechanism, recombinant PigF was purified and crystallized.
Prodigiosin, which is a member of the prodiginines, is a red linear tripyrrole compound. A gene cluster for the biosynthesis of prodigiosin has been identified in Serratia and most genes in the cluster have been functionally assigned. A bifurcated biosynthetic pathway for prodigiosin has previously been determined. The last step in the biosynthetic pathway of 4-methoxy-2,2′-bipyrrole-5-­carbaldehyde (MBC) is catalyzed by PigF, which transfers a methyl group to 4-­hydroxy-2,2′-bipyrrole-5-carbaldehyde (HBC) to form the terminal product MBC, but its catalytic mechanism is not known. To elucidate its mechanism, recombinant PigF was purified and crystallized. The crystals belonged to space group P21, with unit-cell parameters a = 69.4, b = 52.4, c = 279.2 Å, β = 96.8°. The native crystals may contain six molecules in the asymmetric unit, with a V M of 2.17 Å3 Da−1 and a solvent content of 43.43%. A full data set was collected at 2.6 Å resolution using synchrotron radiation on beamline BL17U of Shanghai Synchrotron Radiation Facility (SSRF), People’s Republic of China. Molecular replacement was unsuccessful. To solve the structure of PigF by experimental phasing, selenomethionine-derivativized protein crystals were prepared from a condition with 0.01 M spermidine as an additive. One crystal diffracted to 1.9 Å resolution and a full data set was collected on beamline BL17U at SSRF. The crystal belonged to space group P21, with unit-cell parameters a = 69.0, b = 52.9, c = 93.4 Å, β = 97.3°. Heavy-atom substructure determination and phasing by SAD clearly showed that the crystal contains two molecules in the asymmetric unit, with a V M of 2.19 Å3 Da−1 and a solvent content of 43.82%.
doi:10.1107/S1744309112024001
PMCID: PMC3412768  PMID: 22869117
PigF; O-methyltransferases; prodigiosin synthesis; Serratia
20.  Crystallization and preliminary X-ray diffraction studies of the GhKCH2 motor domain: alteration of pH significantly improved the quality of the crystals 
The GhKCH2 motor domain was crystallized and the pH of the crystallization buffer was shown to have a significant effect on the crystal morphology and diffraction quality.
GhKCH2, a member of the kinesin superfamily, is a plant-specific microtubule-dependent motor protein from cotton with the ability to bind to both microtubules and microfilaments. Here, the motor domain of GhKCH2 (GhKCH2MD; amino acids 371–748) was overexpressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. The pH of the crystallization buffer was shown to have a significant effect on the crystal morphology and diffraction quality. The crystals belonged to space group P212121, with unit-cell parameters a = 60.7, b = 78.6, c = 162.8 Å, α = β = γ = 90°. The Matthews coefficient and solvent content were calculated as 2.27 Å3 Da−1 and 45.87%, respectively. X-ray diffraction data for GhKCH2MD were collected on beamline BL17U1 at Shanghai Synchrotron Radiation Facility and processed to 2.8 Å resolution.
doi:10.1107/S1744309112016351
PMCID: PMC3388925  PMID: 22750868
kinesins; pH; crystal morphology; diffraction quality
21.  Crystallization and preliminary X-ray analysis of a novel halotolerant feruloyl esterase identified from a soil metagenomic library 
A novel feruloyl esterase (EstF27) identified from a soil metagenomic library has been crystallized and a complete data set was collected from a single cooled crystal using an in-house X-ray source.
Feruloyl esterase cleaves the ester linkage formed between ferulic acid and polysaccharides in plant cell walls and thus has wide potential industrial applications. A novel feruloyl esterase (EstF27) identified from a soil metagenomic library was crystallized and a complete data set was collected from a single cooled crystal using an in-house X-ray source. The crystal diffracted to 2.9 Å resolution and belonged to space group P212121, with unit-cell parameters a = 94.35, b = 106.19, c = 188.51 Å, α = β = γ = 90.00°. A Matthews coefficient of 2.55 Å3 Da−1, with a corresponding solvent content of 51.84%, suggested the presence of ten protein subunits in the asymmetric unit.
doi:10.1107/S1744309112017812
PMCID: PMC3388917  PMID: 22750860
feruloyl esterase
22.  Purification, crystallization and preliminary X-ray analysis of nonstructural protein 2 (nsp2) from avian infectious bronchitis virus 
Nonstructural protein 2 from avian infectious bronchitis virus has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.8 Å resolution.
Avian infectious bronchitis virus (IBV) is a member of the group III coronaviruses, which differ from the other groups of coronaviruses in that they do not encode the essential pathogenic factor nonstructural protein 1 (nsp1) and instead start with nsp2. IBV nsp2 is one of the first replicase proteins to be translated and processed in the viral life cycle; however, it has an entirely unknown function. In order to better understand the structural details and functional mechanism of IBV nsp2, the recombinant protein was cloned, overexpressed in Escherichia coli, purified and crystallized. The crystals diffracted to 2.8 Å resolution and belonged to space group P21, with unit-cell parameters a = 57.0, b = 192.3, c = 105.7 Å, β = 90.8°. Two molecules were found in the asymmetric unit; the Matthews coefficient was 3.9 Å3 Da−1, corresponding to a solvent content of 68.2%.
doi:10.1107/S1744309112018623
PMCID: PMC3370919  PMID: 22684079
avian infectious bronchitis virus; nonstructural protein 2
23.  ‘Seeding’ with protease to optimize protein crystallization conditions in in situ proteolysis 
Addition of protease instead of seeds using a robot can be used to optimize the concentration of protease in in situ proteolysis experiments and has been successfully tested using two proteins.
In situ proteolysis is one of the most effective rescue strategies for protein crystallization, and optimization of the ratio between the protein and the protease is one of the key steps in the process. Seeding is a very powerful tool to optimize crystallization conditions and can be performed by most crystallization robots. Addition of protease instead of seed stock using a robot can be used to optimize the concentration of protease in in situ proteolysis experiments and has been successfully tested using two proteins.
doi:10.1107/S174430911201161X
PMCID: PMC3374523  PMID: 22691798
in situ proteolysis; optimization
24.  Crystallization and preliminary X-ray diffraction analysis of the SOD-TAT fusion protein 
A cell membrane permeable SOD, SOD-TAT fusion protein, was expressed, purified and crystallized. X-ray diffraction data have been collected to 3.2 Å resolution.
The superoxide dismutase (SOD) family of proteins are necessary to protect oxygen-utilizing cells from the toxicity of reactive oxygen species. The delivery of SOD into tissues is severely limited by its size and biochemical properties. A cell-membrane-permeable SOD, SOD-TAT, has been demonstrated to have the ability to be directly transduced into mammalian cells. In this study, the SOD-TAT fusion protein was expressed, purified and crystallized. Crystals of the SOD-TAT fusion protein diffracted to 3.20 Å resolution and belonged to space group C121.
doi:10.1107/S1744309112012031
PMCID: PMC3374509  PMID: 22691784
SOD-TAT fusion protein; superoxide dismutases
25.  Preliminary crystallographic analysis of two hypothetical ribose-5-phosphate isomerases from Streptococcus mutans  
Two hypothetical ribose-5-phosphate isomerases from S. mutans have been produced in E. coli and crystallized. The crystals diffracted to high resolutions suitable for crystallographic analyses.
Study of the enzymes from sugar metabolic pathways may provide a better understanding of the pathogenesis of the human oral pathogen Streptococcus mutans. Bioinformatics, biochemical and crystallization methods were used to characterize and understand the function of two putative ribose-5-phosphate isomerases: SMU1234 and SMU2142. The proteins were cloned and constructed with N-terminal His tags. Protein purification was performed by Ni2+-chelating and size-exclusion chromatography. The crystals of SUM1234 diffracted to 1.9 Å resolution and belonged to space group P212121, with unit-cell parameters a = 48.97, b = 98.27, c = 101.09 Å, α = β = γ = 90°. The optimized SMU2142 crystals diffracted to 2.7 Å resolution and belonged to space group P1, with unit-cell parameters a = 53.7, b = 54.1, c = 86.5 Å, α = 74.2, β = 73.5, γ = 83.7°. Initial phasing of both proteins was attempted by molecular replacement; the structure of SMU1234 could easily be solved, but no useful results were obtained for SMU2142. Therefore, SeMet-labelled SMU2142 will be prepared for phasing.
doi:10.1107/S1744309112009761
PMCID: PMC3374514  PMID: 22691789
ribose-5-phosphate isomerase; Streptococcus mutans

Results 1-25 (103)