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1.  Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris  
Diisopropyl fluorophosphatase (DFPase) effectively hydrolyzes a number of organophosphorus nerve agents, including sarin, cyclohexylsarin, soman and tabun. Neutron diffraction data have been collected from DFPase crystals to 2.2 Å resolution in an effort to gain further insight into the mechanism of this enzyme.
The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D2O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2 Å resolution on a relatively small (0.43 mm3) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.
doi:10.1107/S1744309106052924
PMCID: PMC2330113  PMID: 17183172
neutron diffraction; DFPase; time-of-flight; phosphotriesterase
2.  Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris 
The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D2O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2Å resolution on a relatively small (0.43 mm3) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.
doi:10.1107/S1744309106052924
PMCID: PMC2330113  PMID: 17183172
3.  Crystallization of hepatocyte nuclear factor 1β in complex with DNA 
Sample preparation, characterization, crystallization and preliminary X-ray analysis are reported for a HNF1β–DNA binary complex.
Hepatocyte nuclear factor 1β (HNF1β) is a member of the POU transcription-factor family and binds the target DNA as a dimer with nanomolar affinity. The HNF1β–DNA complex has been prepared and crystallized by hanging-drop vapor diffusion in 6%(v/v) PEG 300, 5%(w/v) PEG 8000, 8%(v/v) glycerol and 0.1 M Tris pH 8.0. The crystals diffracted to 3.2 Å (93.9% completeness) using a synchrotron-radiation source under cryogenic (100 K) conditions and belong to space group R3, with unit-cell parameters a = b = 172.69, c = 72.43 Å. A molecular-replacement solution has been obtained and structure refinement is in progress. This structure will shed light on the molecular mechanism of promoter recognition by HNF1β and the molecular basis of the disease-causing mutations found in it.
doi:10.1107/S1744309106015168
PMCID: PMC1581457  PMID: 16754972
protein–DNA complex; diabetes; POU transcription factor
4.  Crystallization of Hepatocyte Nuclear Factor 1β in Complex with DNA 
Hepatocyte nuclear factor 1β (HNF1β) is a member of the POU transcriptionfactor family and binds the target DNA as a dimer with nanomolar affinity. The HNF1β-DNA complex has been prepared and crystallized by hanging-drop vapor diffusion in 6%(v/v) PEG 300, 5%(w/v) PEG 8000, 8%(v/v) glycerol and 0.1 M Tris pH 8.0. The crystals diffracted to 3.2Å (93.9% completeness) using a synchrotron-radiation source under cryogenic (100 K) conditions and belong to space group R3, with unit-cell parameters a = b = 172.69, c = 72.43Å. A molecular-replacement solution has been obtained and structure refinement is in progress. This structure will shed light on the molecular mechanism of promoter recognition by HNF1β and the molecular basis of the disease-causing mutations found in it.
doi:10.1107/S1744309106015168
PMCID: PMC1581457  PMID: 16754972
5.  Expression, purification, crystallization and preliminary crystallographic analysis of BipD, a component of the Burkholderia pseudomallei type III secretion system 
A construct consisting of residues 10–310 of mature BipD, a component of the B. pseudomallei type III secretion system, has been crystallized. Native BipD crystals and SeMet and K2PtCl4 derivative crystals have undergone preliminary crystallographic analysis.
A construct consisting of residues 10–310 of BipD, a component of the Burkholderia pseudomallei type III secretion system (T3SS), has been overexpressed as a GST fusion, cleaved from the GST tag and purified. Crystals were grown of native and selenomethionine-labelled BipD. The crystals grow in two different polymorphs from the same condition. The first polymorph belongs to space group C222, with unit-cell parameters a = 103.98, b = 122.79, c = 49.17 Å, a calculated Matthews coefficient of 2.4 Å3 Da−1 (47% solvent content) and one molecule per asymmetric unit. The second polymorph belongs to space group P21212, with unit-cell parameters a = 136.47, b = 89.84, c = 50.15 Å, and a calculated Matthews coefficient of 2.3 Å3 Da−1 (45% solvent content) for two molecules per asymmetric unit (analysis of the self-rotation function indicates the presence of a weak twofold non-crystallographic symmetry axis in this P21212 form). The native crystals of both forms give diffraction data to 2.7 Å resolution, while the SeMet-labelled P21212 crystals diffract to 3.3 Å resolution. A K2PtCl4 derivative of the P21212 form was also obtained and data were collected to 2.7 Å with radiation of wavelength λ = 0.933 Å. The Pt-derivative anomalous difference Patterson map revealed two self-peaks on the Harker sections.
doi:10.1107/S1744309106027035
PMCID: PMC1894745  PMID: 16946464
BipD; type III secretion system; Burkholderia pseudomallei
6.  Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system 
IpaD, the putative needle-tip protein of the S. flexneri type III secretion system, has been crystallized in a variety of crystal forms using in-drop proteolysis. Native and selenomethionine-labelled data collection and preliminary analyses are reported.
IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P212121, with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 Å, and data were collected to 2.9 Å resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 Å resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 Å, β = 107.9°. An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit.
doi:10.1107/S1744309106027047
PMCID: PMC1894744  PMID: 16946465
IpaD; type III secretion; Shigella flexneri
7.  Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle 
A monodisperse truncation mutant of MxiH, the subunit of the S. flexneri type III secretion system needle, has been crystallized. SeMet derivatives and a uranyl derivative have undergone preliminary crystallographic analysis.
A monodisperse truncation mutant of MxiH, the subunit of the needle from the Shigella flexneri type III secretion system (TTSS), has been overexpressed and purified. Crystals were grown of native and selenomethionine-labelled MxiHCΔ5 and diffraction data were collected to 1.9 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 183.4, b = 28.1, c = 27.8 Å, β = 96.5°. An anomalous difference Patterson map calculated with the data from the SeMet-labelled crystals revealed a single peak on the Harker section v = 0. Inspection of a uranyl derivative also revealed one peak in the isomorphous difference Patterson map on the Harker section v = 0. Analysis of the self-rotation function indicates the presence of a twofold non-crystallographic symmetry axis approximately along a. The calculated Matthews coefficient is 1.9 Å3 Da−1 for two molecules per asymmetric unit, corresponding to a solvent content of 33%.
doi:10.1107/S1744309106006555
PMCID: PMC1894743  PMID: 16511329
MxiH; type III secretion system; Shigella flexneri
8.  Structure of the catalytic domain of the hyperthermophilic chitinase from Pyrococcus furiosus  
The crystal structure of the catalytic domain of a chitinase from P. furiosus is reported.
The crystal structure of the catalytic domain of a chitinase from the hyperthermophilic archaeon Pyrococcus furiosus (AD2PF-ChiA) has been determined at 1.5 Å resolution. This is the first structure of the catalytic domain of an archaeal chitinase. The overall structure of AD2PF-ChiA is a TIM-barrel fold with a tunnel-like active site that is a common feature of family 18 chitinases. Although the catalytic residues (Asp522, Asp524 and Glu526) are conserved, comparison of the conserved residues and structures with those of other homologous chitinases indicates that the catalytic mechanism of PF-ChiA is different from that of family 18 chitinases.
doi:10.1107/S1744309106051773
PMCID: PMC2330115  PMID: 17183162
9.  Preliminary crystallographic analysis of avian infectious bronchitis virus main protease 
The avian infectious bronchitis virus main protease has been crystallized; crystals diffract to 2.7 Å resolution.
Infectious bronchitis virus (IBV) is the prototype of the genus Coronavirus. It causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. The coronavirus (CoV) main protease (Mpro), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. In this study, IBV Mpro was overexpressed in Escherichia coli. Crystals suitable for X-ray crystallography have been obtained using microseeding techniques and belong to space group P6122. X-ray diffraction data were collected in-house to 2.7 Å resolution from a single crystal. The unit-cell parameters were a = b = 119.1, c = 270.7 Å, α = β = 90, γ = 120°. Three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function.
doi:10.1107/S1744309106052341
PMCID: PMC2330114  PMID: 17183167
infectious bronchitis virus; main protease
10.  Effects of the magnesium and chloride ions and shikimate on the structure of shikimate kinase from Mycobacterium tuberculosis  
The crystal structure of MtSK in complex with ADP shows conformational changes caused by the absence of the shikimate molecule, and the effect of chloride ions on the nucleotide binding site, and the crystal structure of MtSK in absence of magnesium shows its effect on the shikimate binding site.
Bacteria, fungi and plants can convert carbohydrate and phosphoenolpyruvate into chorismate, which is the precursor of various aromatic compounds. The seven enzymes of the shikimate pathway are responsible for this conversion. Shikimate kinase (SK) is the fifth enzyme in this pathway and converts shikimate to shikimate-3-phosphate. In this work, the conformational changes that occur on binding of shikimate, magnesium and chloride ions to SK from Mycobacterium tuberculosis (MtSK) are described. It was observed that both ions and shikimate influence the conformation of residues of the active site of MtSK. Magnesium influences the conformation of the shikimate hydroxyl groups and the position of the side chains of some of the residues of the active site. Chloride seems to influence the affinity of ADP and its position in the active site and the opening length of the LID domain. Shikimate binding causes a closing of the LID domain and also seems to influence the crystallographic packing of SK. The results shown here could be useful for understanding the catalytic mechanism of SK and the role of ions in the activity of this protein.
doi:10.1107/S1744309106046823
PMCID: PMC2330112  PMID: 17183161
shikimate
11.  Purification, crystallization and preliminary crystallographic analysis of the putative thiamine-biosynthesis protein PH1313 from Pyrococcus horikoshii OT3 
The putative thiamine-biosynthesis protein PH1313 from P. horikoshii OT3 was overexpressed, purified and crystallized. The crystals belong to space group P212121 and diffract X-rays to 1.9 Å resolution.
The putative thiamine-biosynthesis protein PH1313 from Pyrococcus horikoshii OT3 has been overexpressed and purified. Crystallization was performed by the oil-microbatch method using 28%(v/v) 2-methyl-2,4-pentanediol as a precipitant at 291 K. A native X-ray diffraction data set at 1.9 Å resolution and a single anomalous dispersion data set from a selenomethionine-derivative crystal at 2.1 Å resolution were collected using synchrotron radiation at 100 K. The native crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 71.7, b = 71.2, c = 141.8 Å.
doi:10.1107/S1744309106054509
PMCID: PMC2330111  PMID: 17183176
thiamine; 4-thiouridine; ThiI; Pyrococcus horikoshii
12.  Exploration of triple-helical fragments: crystallization and preliminary X-ray diffraction of d(TGGCCTTAAGG) 
The crystallization and X-ray data collection of the undecamer d(TGGCCTTAAGG) are reported.
The nonamer d(GCGAATTCG) and decamer d(GGCCAATTGG), containing one and two overhanging guanines, respectively, form G·GC triplets in their crystal packing. In order to introduce a third subsequent T·AT triplet, the decamer was further extended by one overhanging thymine residue. Two different crystal morphologies of the sequence d(TGGCCTTAAGG) were obtained by hanging-drop vapour diffusion and diffracted to 2.5 and 2.3 Å resolution, respectively. However, both crystals belong to the orthorhombic space group P212121, with similar unit-cell parameters. Therefore, the two data sets could be merged to a resolution of 2.4 Å with unit-cell parameters a = 26.97, b = 41.12, c = 52.72 Å.
doi:10.1107/S1744309106054522
PMCID: PMC2330110  PMID: 17183175
triple helix; triplet; Hoogsteen; reverse Hoogsteen
13.  Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tails of adhesion molecules CD43 and PSGL-1 
The radixin FERM domain has been crystallized in complex with CD43 and PSGL-1 peptides. Diffraction data sets were collected from the complexes to 2.9 and 2.8 Å resolution, respectively.
Radixin is a member of the ERM proteins that cross-link plasma membranes and actin filaments. The FERM domains located in the N-terminal regions of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. Here, crystals of the radixin FERM domain bound to the cytoplasmic peptides of two adhesion molecules, CD43 and PSGL-1, have been obtained. Crystals of the radixin FERM domain bound to CD43 belong to space group P4322, with unit-cell parameters a = b = 68.72, c = 201.39 Å, and contain one complex in the crystallographic asymmetric unit. Crystals of the radixin FERM domain bound to PSGL-1 belong to space group P212121, with unit-cell parameters a = 80.74, b = 85.73, c = 117.75 Å, and contain two complexes in the crystallographic asymmetric unit. Intensity data sets were collected to a resolution of 2.9 Å for the FERM–CD43 complex and 2.8 Å for the FERM–PSGL-1 complex.
doi:10.1107/S1744309106054145
PMCID: PMC2330108  PMID: 17183174
ERM; CD43; PSGL-1
14.  Preliminary X-ray analysis of XC5848, a hypothetical ORFan protein with an Sm-like motif from Xanthomonas campestris  
A conserved hypothetical protein Se-XC5848 from X. campestris pv. campestris has been overexpressed in E. coli, purified and crystallized. Crystals obtained from the purified recombinant protein diffracted to a resolution of 1.68 Å.
XC5848, a hypothetical protein from the pathogenic bacterium Xanthomonas campestris that causes black rot, has been chosen as a potential target for the discovery of novel folds. It is unique to the Xanthomonas genus and has significant sequence identity mainly to corresponding proteins from the Xanthomonas genus. In this paper, the cloning, overexpression, purification and crystallization of the XC5848 protein are reported. The XC5848 crystals diffracted to a resolution of at least 1.68 Å. They belong to the orthorhombic space group P212121, with unit-cell parameters a = 48.13, b = 51.62, c = 82.32 Å. Two molecules were found in each asymmetric unit. Preliminary structural studies nevertheless indicate that XC5848 belongs to the highly conserved Sm-like α-­β-­β-β-β fold. However, significant differences in sequence and structure were observed. It therefore represents a novel variant of the crucial Sm-like motif that is heavily involved in mRNA splicing and degradation.
doi:10.1107/S1744309106052730
PMCID: PMC2330107  PMID: 17183169
Xanthomonas campestris; structural genomics; conserved hypothetical proteins; ORFans; Sm-like motif
15.  Crystallization and preliminary X-ray diffraction analysis of an Escherichia coli tRNAGly acceptor-stem microhelix 
In order to investigate the identity elements of the E. coli tRNAGly/GlyRS class II system, a tRNAGly acceptor-stem microhelix was crystallized and a data set was collected to 2.0 Å resolution using synchrotron radiation.
The tRNAGly and glycyl-tRNA synthetase (GlyRS) system is an evolutionary special case within the class II aminoacyl-tRNA synthetases because two divergent types of GlyRS exist: an archaebacterial/human type and an eubacterial type. The tRNA identity elements which determine the correct aminoacylation process are located in the aminoacyl domain of tRNAGly. To obtain further insight concerning structural investigation of the identity elements, the Escherichia coli seven-base-pair tRNAGly acceptor-stem helix was crystallized. Data were collected to 2.0 Å resolution using synchrotron radiation. Crystals belong to space group P3121 or P3221, with unit-cell parameters a = b = 35.35, c = 130.82 Å, α = β = 90, γ = 120° and two molecules in the asymmetric unit.
doi:10.1107/S1744309106052870
PMCID: PMC2330105  PMID: 17183173
tRNA acceptor-stem microhelix; tRNA identity elements; tRNAGly; glycyl-tRNA synthetase; class II aminoacyl-tRNA synthetases
16.  Crystallization and preliminary crystallographic analysis of molybdenum-cofactor biosynthesis protein C from Thermus thermophilus  
The molybdenum-cofactor biosynthesis protein C from T. thermophilus has been crystallized in two different space groups, P21 and R32; the crystals diffracted to 1.9 and 1.75 Å resolution, respectively.
The Gram-negative aerobic eubacterium Thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in Japan. The molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. The molybdenum-cofactor biosynthesis protein C derived from T. thermophilus was crystallized in two different space groups. Crystals obtained using the first crystallization condition belong to the monoclinic space group P21, with unit-cell parameters a = 64.81, b = 109.84, c = 115.19 Å, β = 104.9°; the crystal diffracted to a resolution of 1.9 Å. The other crystal form belonged to space group R32, with unit-cell parameters a = b = 106.57, c = 59.25 Å, and diffracted to 1.75 Å resolution. Preliminary calculations reveal that the asymmetric unit contains 12 monomers and one monomer for the crystals belonging to space group P21 and R32, respectively.
doi:10.1107/S1744309106052560
PMCID: PMC2330104  PMID: 17183168
thermophilic microorganisms; Thermus thermophilus; molybdenum-cofactor biosynthesis; MoaC
17.  Purification, crystallization and preliminary X-ray analysis of the BseCI DNA methyltransferase from Bacillus stearothermophilus in complex with its cognate DNA 
The DNA methyltransferase M.BseCI from B. stearothermophilus was crystallized as a complex with its cognate DNA. Crystals belong to space group P6 and diffract to 2.5 Å resolution at a synchrotron source.
The DNA methyltransferase M.BseCI from Bacillus stearothermophilus (EC 2.1.1.72), a 579-amino-acid enzyme, methylates the N6 atom of the 3′ adenine in the sequence 5′-ATCGAT-3′. M.BseCI was crystallized in complex with its cognate DNA. The crystals were found to belong to the hexagonal space group P6, with unit-cell parameters a = b = 87.0, c = 156.1 Å, β = 120.0° and one molecule in the asymmetric unit. Two complete data sets were collected at wavelengths of 1.1 and 2.0 Å to 2.5 and 2.8 Å resolution, respectively, using synchrotron radiation at 100 K.
doi:10.1107/S1744309106051530
PMCID: PMC2330102  PMID: 17183163
DNA-modification enzymes; N6-adenine methylation
18.  Purification, crystallization and preliminary crystallographic characterization of the caspase-recruitment domain of human Nod1 
The caspase-recruitment domain of the cytosolic pathogen receptor Nod1 was crystallized. X-ray diffraction data were collected to 1.9 Å resolution.
The caspase-recruitment domain (CARD) is known to play an important role in apoptosis and inflammation as an essential protein–protein interaction domain. The CARD of the cytosolic pathogen receptor Nod1 was overexpressed in Escherichia coli and purified by affinity chromatography and gel filtration. The purified CARD was crystallized at 277 K using the microseeding method. X-ray diffraction data were collected to 1.9 Å resolution. The crystals belong to space group P31 or P32, with unit-cell parameters a = b = 79.1, c = 80.9 Å. Preliminary analysis indicates that there is one dimeric CARD molecule in the asymmetric unit.
doi:10.1107/S1744309106051955
PMCID: PMC2330101  PMID: 17183166
caspase-recruitment domain; Nod1
19.  Expression, purification and preliminary crystallographic studies on the catalytic region of the nonreceptor tyrosine kinase Fes 
The catalytic domain of human Fes tyrosine kinase has been cloned, expressed, purified and crystallized.
The proto-oncogene tyrosine protein kinase c-fps/fes encodes a structurally unique protein (Fes) of the nonreceptor protein-tyrosine kinase (PTK) family. Its expression has been demonstrated in myeloid haematopoietic cells, vascular endothelial cells and in neurons. In human-derived and murine-derived cell lines, the activated form of this kinase can induce cellular transformation; moreover, it has been shown that Fes is involved in the regulation of cell–cell and cell–matrix interactions mediated by adherens junctions and focal adhesions. The N-terminus of Fes contains the FCH (Fps/Fes/Fer/CIP4 homology) domain, which is unique to the Fes/Fer kinase family. It is followed by three coiled-coil domains and an SH2 (Src-homology 2) domain. The catalytic region (Fes-CR) is located at the C-terminus of the protein. The successful expression, purification and crystallization of the catalytic part of Fes (Fes-CR) are described.
doi:10.1107/S1744309106051682
PMCID: PMC2330100  PMID: 17183165
Fes; tyrosine protein kinases
20.  Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3 
An archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue from P. horikoshii OT3 was overexpressed as native and selenomethionine-substituted protein, purified and crystallized. The native and selenomethionine-derivative crystals are isomorphous and diffract X-rays to 2.1 and 2.9 Å resolution, respectively.
6-Pyruvoyl tetrahydrobiopterin synthase (PTPS) catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. PH0634, a 13.51 kDa archaeal PTPS homologue from Pyrococcus horikoshii OT3, was overexpressed as native and selenomethionine-substituted protein and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data were collected to 2.1 Å resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 Å. Threefold noncrystallographic symmetry was identified from self-rotation calculations. Assuming the presence of a trimer in the asymmetric unit, the solvent content is 45% (V M = 2.24 Å3 Da−1). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 Å.
doi:10.1107/S1744309106051578
PMCID: PMC2330099  PMID: 17183164
tetrahydrobiopterin biothynthesis; 6-pyruvoyl tetrahydrobiopterin synthase; Pyrococcus horikoshii OT3
21.  Preliminary structural studies of the transcriptional regulator CmeR from Campylobacter jejuni  
The transcriptional regulator CmeR from C. jejuni has been purified and crystallized and X-ray diffraction data have been collected to a resolution of 2.2 Å.
In Campylobacter jejuni, a Gram-negative bacterial pathogen causing gastroenteritis in humans, the CmeR regulatory protein controls transcription of the multidrug transporter gene operon cmeABC. CmeR belongs to the TetR family of transcriptional regulators. The 210-residue CmeR consists of two functional motifs: an N-terminal DNA-binding domain and a C-terminal ligand-binding domain. It is predicted that the DNA-binding domain interacts directly with target promoters, while the C-terminal motif interacts with inducing ligands (such as bile salts). As an initial step towards confirming this structural model, recombinant CmeR protein containing a 6×His tag at the N-terminus was crystallized. Crystals of ligand-free CmeR belonged to space group P21212, with unit-cell parameters a = 37.4, b = 57.6, c = 93.3 Å. Diffraction was observed to at least 2.2 Å at 100 K. Analysis of the detailed CmeR structure is currently in progress.
doi:10.1107/S1744309106053127
PMCID: PMC2330109  PMID: 17183170
CmeR; transcriptional regulators; gene regulation; multi-ligand recognition
22.  The molecular structure of Rv1873, a conserved hypothetical protein from Mycobacterium tuberculosis, at 1.38 Å resolution 
SIRAS phasing techniques have been employed to determine the structure Mycobacterium tuberculosis Rv1873 at a resolution of 1.38 Å. We show that this conserved hypothetical protein of unknown function shows limited structural similarity to other molecules.
The X-ray crystal structure of the gene product encoded by open reading frame Rv1873 of Mycobacterium tuberculosis has been determined by single isomorphous replacement with anomalous scattering (SIRAS) phasing tech­niques at 1.38 Å resolution from monoclinic crystals with unit-cell parameters a = 33.44, b = 31.63, c = 53.19 Å, β = 90.8°. The 16.2 kDa Rv1873 is a monomer that adopts a primarily α-helical fold with limited structural similarity to previously determined tertiary structures. It has been annotated as a conserved hypothetical protein of unknown function and is classified by the Clusters of Orthologous Groups (COG) database as belonging to COG5579. The three-dimensional structure of the Rv1873 gene product reveals limited similarity to a repeated motif that is found in a variety of other proteins. While not a novel fold, it serves as a model for orthologues predicted to be related by sequence and it is hoped that knowledge of the structure of Rv1873 will aid in determining a possible function for this protein.
doi:10.1107/S1744309106046902
PMCID: PMC2225384  PMID: 17142896
Rv1873; Mycobacterium tuberculosis; COG 5579; DALI; α-karyopherin; ATP synthase
23.  The structure of Pyrococcus horikoshii 2′-5′ RNA ligase at 1.94 Å resolution reveals a possible open form with a wider active-site cleft 
The crystal structure of the 2′-5′ RNA ligase PH0099 from P. horikoshii OT3 was solved at 1.94 Å resolution. The molecule has a bilobal α+β arrangement with two antiparallel β-sheets constituting a V-shaped active-site cleft, as found in other members of the 2H phosphoesterase superfamily.
Bacterial and archaeal 2′-5′ RNA ligases, members of the 2H phosphoesterase superfamily, catalyze the linkage of the 5′ and 3′ exons via a 2′-5′-phosphodiester bond during tRNA-precursor splicing. The crystal structure of the 2′-5′ RNA ligase PH0099 from Pyrococcus horikoshii OT3 was solved at 1.94 Å resolution (PDB code 1vgj). The molecule has a bilobal α+β arrangement with two antiparallel β-sheets constituting a V-shaped active-site cleft, as found in other members of the 2H phosphoesterase superfamily. The present structure was significantly different from that determined previously at 2.4 Å resolution (PDB code 1vdx) in the active-site cleft; the entrance to the cleft is wider and the active site is easily accessible to the substrate (RNA precursor) in our structure. Structural comparison with the 2′-5′ RNA ligase from Thermus thermophilus HB8 also revealed differences in the RNA precursor-binding region. The structural differences in the active-site residues (tetrapeptide motifs H-X-T/S-X) between the members of the 2H phosphoesterase superfamily are discussed.
doi:10.1107/S1744309106046616
PMCID: PMC2225383  PMID: 17142895
2′-5′ RNA ligase; 2H phosphoesterase superfamily; Pyrococcus horikoshii
24.  Cloning, expression, purification, crystallization and preliminary crystallographic analysis of pseudo death-effector domain of HIPPI, a molecular partner of Huntingtin-interacting protein HIP-1 
A pseudo death-effector domain (pDED) of HIPPI, a partner of Huntingtin-interacting protein HIP1, has been cloned, overexpressed and crystallized. The crystals of pDED-HIPPI diffracted to 2.2 Å.
The formation of a heterodimer between Huntingtin-interacting protein-1 (HIP-­1) and its novel partner HIPPI (HIP-1 protein interactor) through their pseudo death-effector domains (pDEDs) is a key step that recruits caspase-8 and initiates apoptosis. This could be one of the pathways by which apoptosis is increased in Huntington’s disease (HD). A construct consisting of the pDED of HIPPI has been cloned and overexpressed as 6NH-tagged protein and purified by Ni–NTA affinity chromatography. Crystals of the pDED of HIPPI were grown in space group P41, with unit-cell parameters a = b = 77.42, c = 33.31 Å and a calculated Matthews coefficient of 1.88 Å3 Da−1 (33% solvent content) with two molecules per asymmetric unit.
doi:10.1107/S1744309106046628
PMCID: PMC2225382  PMID: 17142908
Huntington’s disease; apoptosis; pseudo death-effector domain
25.  Purification, crystallization and preliminary X-ray diffraction studies of UDP-N-acetylglucosamine pyrophosphorylase from Candida albicans  
UDP-N-acetylglucosamine pyrophosphorylase was purified and crystallized and X-ray diffraction data were collected to 2.3 Å resolution.
UDP-N-acetylglucosamine pyrophosphorylase (UAP) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine. UAP from Candida albicans was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals of the substrate and product complexes both diffract X-rays to beyond 2.3 Å resolution using synchrotron radiation. The crystals of the substrate complex belong to the triclinic space group P1, with unit-cell parameters a = 47.77, b = 62.89, c = 90.60 Å, α = 90.01, β = 97.72, γ = 92.88°, whereas those of the product complex belong to the orthorhombic space group P212121, with unit-cell parameters a = 61.95, b = 90.87, c = 94.88 Å.
doi:10.1107/S1744309106044186
PMCID: PMC2225379  PMID: 17142897
UDP-N-acetylglucosamine pyrophosphorylase; Candida albicans

Results 1-25 (344)