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1.  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 
Acta Crystallographica Section F  2006;62(Pt 12):1196-1200.
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
2.  The copper-containing amine oxidase from Arthrobacter globiformis: refinement at 1.55 and 2.20 Å resolution in two crystal forms 
Acta Crystallographica Section F  2006;62(Pt 11):1052-1057.
The previously reported structure of the copper-containing amine oxidase from A. globiformis has been refined in two different crystal forms at resolutions of 1.55 and 2.20 Å.
Copper-containing amine oxidases are found in all the major kingdoms of life. They catalyse the oxidation of organic amines in the presence of molecular dioxygen to aldehydes and hydrogen peroxide. The catalytic centres contain a Cu atom and a topaquinone cofactor formed autocatalytically from a tyrosine residue in the presence of Cu and molecular oxygen. The structure of the Cu-containing amine oxidase from Arthrobacter globiformis, which was previously refined at 1.8 Å resolution in space group C2 with unit-cell parameters a = 157.84, b = 63.24, c = 91.98 Å, β = 112.0° [Wilce et al. (1997 ▶), Biochemistry, 36, 16116–16133], has been re-refined with newly recorded data at 1.55 Å resolution. The structure has also been solved and refined at 2.2 Å resolution in a new crystal form, space group C2, with unit-cell parameters a = 158.04, b = 64.06, c = 69.69 Å, β = 111.7°.
doi:10.1107/S1744309106038814
PMCID: PMC2225227  PMID: 17077478
copper amine oxidase; topaquinone; Arthrobacter globiformis
3.  Structure of ribose 5-phosphate isomerase from Plasmodium falciparum  
Acta Crystallographica Section F  2006;62(Pt 5):427-431.
The crystal structure of ribose 5-phosphate isomerase from P. falciparum has been determined at 2.09 Å resolution. The structure is very similar to those of RpiA from other species, but is in some ways more similar to bacterial homologs than to those from higher eukaryotes.
The structure of ribose 5-phosphate isomerase from Plasmodium falciparum, PFE0730c, has been determined by molecular replacement at 2.09 Å resolution. The enzyme, which catalyzes the isomerization reaction that interconverts ribose 5-phosphate and ribulose 5-phosphate, is a member of the pentose phosphate pathway. The P. falciparum enzyme belongs to the ribose 5-­phosphate isomerase A family, Pfam family PF06562 (DUF1124), and is structurally similar to other members of the family.
doi:10.1107/S1744309106010876
PMCID: PMC2219984  PMID: 16682767
ribose 5-phosphate isomerase
4.  Structure of the conserved hypothetical protein MAL13P1.257 from Plasmodium falciparum  
Acta Crystallographica Section F  2006;62(Pt 3):180-185.
The crystal structure of a conserved hypothetical protein, MAL13P1.257 from P. falciparum, has been determined at 2.17 Å resolution. The structure represents a new protein fold and is the first structural representative for Pfam sequence family PF05907.
The structure of a conserved hypothetical protein, PlasmoDB sequence MAL13P1.257 from Plasmodium falciparum, Pfam sequence family PF05907, has been determined as part of the structural genomics effort of the Structural Genomics of Pathogenic Protozoa consortium. The structure was determined by multiple-wavelength anomalous dispersion at 2.17 Å resolution. The structure is almost entirely β-sheet; it consists of 15 β-strands and one short 310-helix and represents a new protein fold. The packing of the two monomers in the asymmetric unit indicates that the biological unit may be a dimer.
doi:10.1107/S1744309106005847
PMCID: PMC2197184  PMID: 16511296
MAL13P1.257
5.  The nucleotide-binding site of Aquifex aeolicus LpxC 
Acta Crystallographica Section F  2006;62(Pt 11):1082-1086.
The structure of UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in complex with UDP is reported. The complex allows for a description of how the enzyme recognizes and binds a nucleotide moiety and enables the construction of an LpxC-substrate model.
The structure of recombinant Aquifex aeolicus UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in complex with UDP has been determined to a resolution of 2.2 Å. Previous studies have characterized the binding sites of the fatty-acid and sugar moieties of the substrate, UDP-(3-O-hydroxymyristoyl)-N-­acetylglucosamine, but not that of the nucleotide. The uracil-binding site is constructed from amino acids that are highly conserved across species. Hydrophobic associations with the Phe155 and Arg250 side chains in combination with hydrogen-bonding interactions with the main chain of Glu154 and the side chains of Tyr151 and Lys227 position the base. The phosphate and ribose groups are directed away from the active site and interact with Arg137, Lys156, Glu186 and Arg250. The orientation of the phosphate-ribose tail is not conducive to catalysis, perhaps owing to the position of an inhibitory Zn2+. However, based on the position of uracil revealed in this study and on the previously reported complex of LpxC with an inhibitor, a model is proposed for substrate binding.
doi:10.1107/S1744309106041893
PMCID: PMC2225228  PMID: 17077484
lipid A; Aquifex aeolicus; LpxC
6.  Structure of the heterotrimeric PCNA from Sulfolobus solfataricus  
Acta Crystallographica Section F  2006;62(Pt 10):944-948.
The structure of the heterotrimeric PCNA complex from S. sulfataricus is reported to 2.3 Å.
PCNA is a ring-shaped protein that encircles DNA, providing a platform for the association of a wide variety of DNA-processing enzymes that utilize the PCNA sliding clamp to maintain proximity to their DNA substrates. PCNA is a homotrimer in eukaryotes, but a heterotrimer in crenarchaea such as Sulfolobus solfataricus. The three proteins are SsoPCNA1 (249 residues), SsoPCNA2 (245 residues) and SsoPCNA3 (259 residues). The heterotrimeric protein crystallizes in space group P21, with unit-cell parameters a = 44.8, b = 78.8, c = 125.6 Å, β = 100.5°. The crystal structure of this heterotrimeric PCNA molecule has been solved using molecular replacement. The resulting structure to 2.3 Å sheds light on the differential stabilities of the interactions observed between the three subunits and the specificity of individual subunits for partner proteins.
doi:10.1107/S1744309106034075
PMCID: PMC2225174  PMID: 17012780
PCNA; Sulfolobus solfataricus
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

Results 1-25 (344)