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1.  Structural Studies of an Anti-Inflammatory Lectin from Canavalia boliviana Seeds in Complex with Dimannosides 
PLoS ONE  2014;9(5):e97015.
Plant lectins, especially those purified from species of the Leguminosae family, represent the best-studied group of carbohydrate-binding proteins. Lectins purified from seeds of the Diocleinae subtribe exhibit a high degree of sequence identity notwithstanding that they show very distinct biological activities. Two main factors have been related to this feature: variance in key residues influencing the carbohydrate-binding site geometry and differences in the pH-dependent oligomeric state profile. In this work, we have isolated a lectin from Canavalia boliviana (Cbol) and solved its x-ray crystal structure in the unbound form and in complex with the carbohydrates Man(α1-3)Man(α1-O)Me, Man(α1-4)Man(α1-O)Me and 5-bromo-4-chloro-3-indolyl-α-D-mannose. We evaluated its oligomerization profile at different pH values using Small Angle X-ray Scattering and compared it to that of Concanavalin A. Based on predicted pKa-shifts of amino acids in the subunit interfaces we devised a model for the dimer-tetramer equilibrium phenomena of these proteins. Additionally, we demonstrated Cbol anti-inflammatory properties and further characterized them using in vivo and in vitro models.
doi:10.1371/journal.pone.0097015
PMCID: PMC4035259  PMID: 24865454
2.  Crystallization and preliminary X-ray diffraction analysis of the lectin from Dioclea rostrata Benth seeds 
D. rostrata lectin was crystallized by hanging-drop vapor diffusion. The crystal belongs to the orthorhombic space group I222 and diffracted to 1.87 Å resolution.
Lectins from the Diocleinae subtribe (Leguminosae) are highly similar proteins that promote various biological activities with distinctly differing potencies. The structural basis for this experimental data is not yet fully understood. Dioclea rostrata lectin was purified and crystallized by hanging-drop vapour diffusion at 293 K. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 61.51, b = 88.22, c = 87.76 Å.  Assuming the presence of one monomer per asymmetric unit, the solvent content was estimated to be about 47.9%. A complete data set was collected at 1.87 Å resolution.
doi:10.1107/S1744309106001801
PMCID: PMC2150952  PMID: 16511292
lectins; Dioclea rostrata
3.  New crystal forms of Diocleinae lectins in the presence of different dimannosides 
The crystallization and preliminary X-­ray data of Canavalia gladiata lectin (CGL) and C. maritima lectin (CML) complexed with Man(α1-2)Man(α1)OMe, Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe in two crystal forms [the complexes with Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe crystallized in space group P32 and those with Man(α1-2)Man(α1)OMe crystallized in space group I222], which differed from those of the native proteins (P21212 for CML and C222 for CGL), are reported.
Studying the interactions between lectins and sugars is important in order to explain the differences observed in the biological activities presented by the highly similar proteins of the Diocleinae subtribe. Here, the crystallization and preliminary X-­ray data of Canavalia gladiata lectin (CGL) and C. maritima lectin (CML) complexed with Man(α1-2)Man(α1)OMe, Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe in two crystal forms [the complexes with Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe crystallized in space group P32 and those with Man(α1-2)Man(α1)OMe crystallized in space group I222], which differed from those of the native proteins (P21212 for CML and C222 for CGL), are reported. The crystal complexes of ConA-like lectins with Man(α1-4)Man(α1)OMe are reported here for the first time.
doi:10.1107/S1744309106038887
PMCID: PMC2225211  PMID: 17077488
lectin–sugar interactions; Dioocleinae lectins
4.  Crystallization and preliminary X-ray diffraction analysis of a lectin from Canavalia maritima seeds 
A lectin from C. maritima was crystallized using the vapour-diffusion method and crystals diffracted to 2.1 Å resolution. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%, refinement is in progress.
A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 Å resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P21212, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 Å. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way.
doi:10.1107/S1744309104029197
PMCID: PMC1952371  PMID: 16508099
lectins; Canavalia maritima
5.  Overexpression, purification, crystallization and preliminary structural studies of catabolic ornithine transcarbamylase from Lactobacillus hilgardii  
The catabolic ornithine transcarbamylase (cOTC) from L. hilgardii has been overexpressed in E. coli, purified and crystallized under two different experimental conditions. The structure has been solved by the molecular-replacement method using the atomic coordinates of catabolic ornithine transcarbamylase from P. aeruginosa as the search model.
The catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) from the lactic acid bacteria Lactobacillus hilgardii is a key protein involved in the degradation of arginine during malolactic fermentation. cOTC containing an N-terminal His6 tag has been overexpressed in Escherichia coli, purified and crystallized under two different experimental conditions using the hanging-drop vapour-diffusion method. Crystals obtained from a solution containing 8%(w/v) PEG 4000, 75 mM sodium acetate pH 4.6 belong to the trigonal space group P321 and have unit-cell parameters a = b = 157.04, c = 79.28 Å. Conversely, crystals grown in 20%(v/v) 2-methyl-2,4-pentanediol, 7.5%(w/v) PEG 4000, 100 mM HEPES pH 7.8 belong to the monoclinic space group C2 and have unit-cell parameters a = 80.06, b = 148.90, c = 91.67 Å, β = 100.25°. Diffraction data were collected in-house to 3.00 and 2.91 Å resolution for trigonal and monoclinic crystals, respectively. The estimated Matthews coefficient for the crystal forms were 2.36 and 2.24 Å3 Da−1, respectively, corresponding to 48% and 45% solvent content. In both cases, the results are consistent with the presence of three protein subunits in the asymmetric unit. The structure of cOTC has been determined by the molecular-replacement method using the atomic coordinates of cOTC from Pseudomonas aeruginosa (PDB code 1dxh) as the search model.
doi:10.1107/S1744309107025195
PMCID: PMC2335135  PMID: 17620711
catabolic ornithine transcarbamylase; Lactobacillus hilgardii; arginine degradation
6.  Crystallization and preliminary X-ray crystallographic study of a putative aspartyl-tRNA synthetase from the crenarchaeon Sulfolobus tokodaii strain 7 
A putative nondiscriminating aspartyl-tRNA synthetase from the crenarchaeon S. tokodaii strain 7 has been recombinantly expressed, purified and crystallized. The crystal structure has been preliminarily solved at 2.3 Å resolution by the molecular-replacement method.
Genome analysis suggests that the aspartyl-tRNA synthetase of the crenarchaeon Sulfolobus tokodaii strain 7 belongs to the nondiscriminating type that is believed to catalyze aspartylation of tRNAAsp and tRNAAsn. This protein has been overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method from 100 mM sodium HEPES buffer pH 7.5 containing 100 mM NaCl and 1.6 M (NH4)2SO4 as the crystallizing reagent. Diffraction data were collected to 2.3 Å resolution using synchrotron radiation. The crystal belongs to the orthorhombic space group P21212, with unit-cell parameters a = 116.0, b = 139.3, c = 75.3 Å. The estimated Matthews coefficient (3.10 Å3 Da−1; 60.3% solvent content) suggests the presence of two subunits in the asymmetric unit. The structure has been successfully solved by the molecular-replacement method. Full refinement of the structure may reveal it to be the original ancestor of the nondiscriminating AspRS.
doi:10.1107/S1744309107026905
PMCID: PMC2335148  PMID: 17620724
aspartyl-tRNA synthetases; Sulfolobus tokodaii strain 7
7.  Purification, crystallization and preliminary X-ray diffraction studies on avian haemoglobin from pigeon (Columba livia) 
Crystallization of pigeon haemoglobin at low pH (5.5) and high ionic concentration (1 M) using the hanging-drop vapour-diffusion method is reported.
Haemoglobin is a physiologically significant metalloprotein that is involved in the exchange of gases for sustaining life. The respiratory system of birds is unique and complex compared with that of mammals. Many investigations of avian haemoglobins have revealed the presence of inositol pentaphosphate (IP5), a principal allosteric effector that is involved in regulation of their function. Structural investigations of avian haemoglobins are presently not adequate to explain their function. Efforts have been made in this direction in order to understand the oxygen-binding affinity involved in adapting to hypoxia in avian haemoglobins. Fresh whole blood was collected from pigeon (Columba livia) and purified using a DEAE cellulose anion-exchange chromatographic column. Crystallization of pigeon haemoglobin was accomplished using the hanging-drop vapour-diffusion method using PEG 3350 as a precipitant in 50 mM sodium acetate buffer pH 5.5 with 1 M NaCl. Data collection was carried out using a MAR345 image-plate detector system. The crystals diffracted to 2 Å resolution. Pigeon haemoglobin crystallizes in a triclinic space group, with two whole biological molecules in the asymmetric unit and with unit-cell parameters a = 55.005, b = 65.528, c = 104.370 Å, α = 78.742, β = 89.819, γ = 65.320°.
doi:10.1107/S1744309108038505
PMCID: PMC2635874  PMID: 19194000
allosteric effectors; oxygen affinity; triclinic; avian haemoglobins
8.  Crystallization and preliminary X-ray diffraction analysis of HML, a lectin from the red marine alga Hypnea musciformis  
The crystallization and preliminary X-ray diffraction analysis of a red marine alga lectin isolated from H. musciformis is reported.
HML, a lectin from the red marine alga Hypnea musciformis, defines a novel lectin family. Orthorhombic crystals of HML belonging to space group P212121 grew within three weeks at 293 K using the hanging-drop vapour-diffusion method. A complete data set was collected at 2.4 Å resolution. HML is the first marine alga lectin to be crystallized.
doi:10.1107/S1744309105033671
PMCID: PMC1978131  PMID: 16511217
red marine algal lectin; Hypnea musciformis; novel lectin family
9.  Expression, purification and preliminary crystallographic analysis of human thyroid hormone responsive protein 
Human Thrsp has been crystallized as a prelude to the determination of its three-dimensional structure by X-ray crystallography.
Thyroid hormone responsive protein (Thrsp, also known as Spot 14 and S14) is a carbohydrate-inducible and thyroid-hormone-inducible nuclear protein specific to liver, adipose and lactating mammary tissues. Thrsp functions to activate genes encoding fatty-acid synthesis enzymes. Recent studies have shown that in some cancers human Thrsp (hS14) localizes to the nucleus and is amplified, suggesting that it plays a role in the regulation of lipogenic enzymes during tumourigenesis. Thrsp, a member of the Spot 14 superfamily, is an acidic homodimeric protein with no sequence similarity to other mammalian gene products and its biochemical function is elusive. To shed light on the structure–function relationship of this protein, human Thrsp was crystallized. Recombinant human Thrsp (hThrsp), the N-terminally truncated human Thrsp10–146 (hThrsp9) and their selenomethionyl (SeMet) derivatives were expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Diffraction-quality crystals were grown at 293 K using Li2SO4 as a precipitant. Using synchrotron radiation, data for the hThrsp SeMet derivative, hThrsp9 and its SeMet derivative were collected to 4.0, 3.0 and 3.6 Å resolution, respectively, at 100 K. The crystals of full-length hThrsp and its SeMet derivative belonged to space group P41212, with approximate unit-cell parameters a = b = 123.9, c = 242.1 Å, α = β = γ = 90.0°. In contrast, the crystals of the truncated hThrsp9 and its SeMet derivative belonged to space group P212121, with approximate unit-cell parameters a = 91.6, b = 100.8, c = 193.7 Å, α = β = γ = 90.0°. A molecular-replacement solution calculated using a murine Spot 14 structure as a search model indicated the presence of six molecules per asymmetric unit, comprising three hThrsp homodimers.
doi:10.1107/S1744309111021099
PMCID: PMC3151134  PMID: 21821901
breast cancer; lipogenesis; Spot 14; S14; Thrsp
10.  Crystallization and X-ray diffraction analysis of nylon-oligomer hydrolase (NylC) from Agromyces sp. KY5R 
Crystals of 6-aminohexanoate-oligomer hydrolase have been obtained by the sitting-drop vapour-diffusion method using sodium citrate as a precipitant. Diffraction data for native and K2PtCl4-derivative crystals were collected to resolutions of 2.00 and 2.20 Å, respectively.
6-Aminohexanoate-oligomer hydrolase (NylC) from Agromyces sp. KY5R was expressed in Escherichia coli JM109 and purified by ammonium sulfate fractionation, anion-exchange column chromatography and gel-filtration chromatography. NylC was crystallized by the sitting-drop vapour-diffusion method with sodium citrate as a precipitant in 0.1 M HEPES buffer pH 7.5 containing 0.2 M NaCl. Diffraction data were collected from native and K2PtCl4-derivative crystals to resolutions of 2.00 and 2.20 Å, respectively. The obtained crystal was plate-shaped, with an I-centred orthorhombic space group and unit-cell parameters a = 155.86, b = 214.45, c = 478.80 Å. The anomalous difference Patterson map of the K2PtCl4-derivative crystal suggested that the space group was I222 rather than I212121.
doi:10.1107/S1744309111022858
PMCID: PMC3151121  PMID: 21821888
6-aminohexanoate-oligomer hydrolase; Agromyces sp. KY5R
11.  Crystallization and preliminary X-ray characterization of a lectin from Cicer arietinum (chickpea) 
The crystallization and characterization of a lectin isolated and purified from C. arietinum and possessing complex sugar specificity is reported.
The lectin isolated from mature seeds of Cicer arietinum (CAL) agglutinates pronase-treated rabbit and human erythrocytes and its haemagglutination activity is inhibited by fetuin and desialated fetuin but not by simple monosaccharides or oligosaccharides. The purified lectin is a dimer of molecular weight 43 000 Da composed of two identical subunits (MW 21 500), as confirmed by SDS–PAGE. The lectin has been crystallized using the hanging-drop vapour-diffusion method at 295 K over a well solution containing 0.2 M sodium acetate, 0.1 M sodium phosphate buffer pH 6.5 and 14%(w/v) polyethylene glycol 8000. The triangular prism-shaped crystals belong to space group R3 and have unit-cell parameters a = b = 81.2, c = 69.4 Å. The diffraction data are 93.8% complete to 2.3 Å Bragg spacing with an R merge of 0.103.
doi:10.1107/S1744309104032166
PMCID: PMC1952404  PMID: 16508116
complex sugar specificity; legume lectin; seed albumins
12.  Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus 
The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described.
White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-­terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P212121, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution.
doi:10.1107/S1744309107029351
PMCID: PMC2335124  PMID: 17620728
white spot syndrome virus; envelope proteins
13.  Crystallization and identification of the glycosylated moieties of two isoforms of the main allergen Hev b 2 and preliminary X-­ray analysis of two polymorphs of isoform II 
Crystallization of important glycoenzymes involved in IgE-mediated latex allergy.
Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a β-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content constisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapour-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P41 with unit-cell parameters a = b = 150.17, c = 77.41 Å were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 Å, β = 113.6°. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.
doi:10.1107/S1744309107039838
PMCID: PMC2376315  PMID: 17768356
Hevea brasiliensis; latex allergens; endo-β-1,3-glucanases; plant glycans
14.  Purification, crystallization and preliminary X-ray analysis of a hexameric β-glucosidase from wheat 
Recombinant β-glucosidase from wheat seedlings complexed with a substrate aglycone has been crystallized in a hexameric active form. A diffraction data set has been collected at 1.7 Å.
The wheat β-glucosidase TaGlu1b, which is only active in a hexameric form, was tagged with 6×His at the N-terminus, overexpressed in Escherichia coli and purified in two steps. The protein complexed with a substrate aglycone was crystallized at 293 K from a solution containing 10 mM HEPES pH 7.2, 1 M LiSO4 and 150 mM NaCl using the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.7 Å at the Photon Factory. The crystal belongs to space group P4132, with unit-cell parameters a = b = c = 194.65 Å, α = β = γ = 90°. The asymmetric unit was confirmed by molecular-replacement solution to contain one monomer, giving a solvent content of 72.1%.
doi:10.1107/S1744309105027028
PMCID: PMC1978116  PMID: 16511181
β-glucosidases; DIMBOA; hexamers; wheat
15.  Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis 
Raucaffricine glucosidase, an enzyme involved in the biosynthesis of monoterpenoid indole alkaloids in the plant Rauvolfia serpentina, was crystallized by the hanging-drop vapour-diffusion method using PEG4000 as precipitant. The crystals diffract to 2.3 Å resolution and belong to space group I222.
Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 Å, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 Å.
doi:10.1107/S174430910600457X
PMCID: PMC2197181  PMID: 16511316
raucaffricine glucosidase; indole alkaloid metabolism; biosynthesis; Rauvolfia serpentina
16.  Crystallization and preliminary crystallographic analysis of NAD+-preferring aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum  
NAD+-preferring aldohexose dehydrogenase from the thermoacidophilic archaeon T. acidophilum was crystallized using the hanging-drop vapour-diffusion technique and X-ray diffraction data were collected to a resolution of 2.8 Å.
The aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a 28 kDa molecular-weight enzyme that catalyzes the oxidation of various aldohexoses, with a preference for NAD+ rather than NADP+ as a cofactor. The recombinant AldT was crystallized using the hanging-drop vapour-diffusion technique at 293 K under several acidic conditions with polyethylene glycol (PEG) and ammonium sulfate as precipitants. Optimization of the initial crystallizations conditions yielded single crystals in solution containing 0.1 M sodium acetate pH 4.6, 18%(w/v) PEG 4000, 0.2 M ammonium sulfate and 15%(v/v) glycerol. An X-ray diffraction data set was collected to a resolution of 2.8 Å.
doi:10.1107/S1744309106017362
PMCID: PMC2243078  PMID: 16754989
aldohexose dehydrogenase; glucose dehydrogenase; d-mannose; short-chain dehydrogenase/reductase (SDR) family; Thermoplasma acidophilum
17.  Purification, crystallization and preliminary crystallographic analysis of very-long-chain acyl-­CoA dehydrogenase from Caenorhabditis elegans  
Very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans (cVLCAD) has been crystallized in space group C2 and its X-ray diffraction data set has been collected to 1.6 Å resolution. Unlike other VLCADs that were reported to form dimers, the purified cVLCAD was found as a homotetrameric protein according to static light-scattering measurements.
Acyl-CoA dehydrogenase [acyl-CoA:(acceptor) 2,3-oxidoreductase; EC 1.3.99.3] catalyzes the first reaction step in mitochondrial fatty-acid β-oxidation. Here, the very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans (cVLCAD) has been cloned and overexpressed in Escherichia coli strain BL21 (DE3). Interestingly, unlike other very-long-chain acyl-CoA dehydrogenases, cVLCAD was found to form a tetramer by size-exclusion chromatography coupled with in-line static light-scattering, refractive-index and ultraviolet measurements. Purified cVLCAD (12 mg ml−1) was successfully crystallized by the hanging-drop vapour-diffusion method under conditions containing 100 mM Tris–HCl pH 8.0, 150 mM sodium chloride, 200 mM magnesium formate and 13% PEG 3350. The crystal has a tetragonal form and a complete diffraction data set was collected and processed to 1.8 Å resolution. The crystal belonged to space group C2, with unit-cell parameters a = 138.6, b = 116.7, c = 115.3 Å, α = γ = 90.0, β = 124.0°. A self-rotation function indicated the existence of one noncrystallographic twofold axis. A preliminary molecular-replacement solution further confirmed the presence of two molecules in one asymmetric unit, which yields a Matthews coefficient V M of 2.76 Å3 Da−1 and a solvent content of 55%.
doi:10.1107/S1744309110005002
PMCID: PMC2852336  PMID: 20383014
very-long-chain acyl-CoA dehydrogenases; fatty-acid β-oxidation; Caenorhabditis elegans; SEC-LS/UV/RI
18.  Crystallization and preliminary X-ray diffraction studies of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus P1 
A hyperthermophilic archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from S. solfataricus P1 has been crystallized as a recombinant protein with a vector-derived long N-terminal extension region. The P43212 crystals of recombinant ARF diffracted to 1.85 Å resolution using synchrotron radiation.
The hyperthermophilic archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 contains a low-potential Rieske-type [2Fe–2S] cluster that has served as a tractable model for ligand-substitution studies on this protein family. Recombinant ARF harbouring a pET30a vector-derived N-­terminal extension region plus a hexahistidine tag has been heterologously overproduced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using 0.05 M sodium acetate, 0.05 M HEPES, 2 M ammonium sulfate pH 5.5. The crystals diffracted to 1.85 Å resolution and belonged to the tetragonal space group P43212, with unit-cell parameters a = 60.72, c = 83.31 Å. The asymmetric unit contains one protein molecule.
doi:10.1107/S1744309110019263
PMCID: PMC2898476  PMID: 20606288
Rieske-type ferredoxins; [2Fe–2S] clusters; archaea; Sulfolobus solfataricus
19.  Crystallization screening test for the whole-cell project on Thermus thermophilus HB8 
Three conventional robots were subjected to a crystallization screening test involving 18 proteins from T. thermophilus HB8 using the sitting- and hanging-drop vapour-diffusion and microbatch methods. The number of diffraction-quality crystals and the amount of time required to obtain visible crystals depended greatly on the robots used. The combined use of different robots, especially for protein samples exhibiting low crystallization success rates, significantly increased the chance of obtaining diffraction-quality crystals.
It was essential for the structural genomics of Thermus thermophilus HB8 to efficiently crystallize a number of proteins. To this end, three conventional robots, an HTS-80 (sitting-drop vapour diffusion), a Crystal Finder (hanging-drop vapour diffusion) and a TERA (modified microbatch) robot, were subjected to a crystallization condition screening test involving 18 proteins from T. thermophilus HB8. In addition, a TOPAZ (microfluidic free-interface diffusion) designed specifically for initial screening was also briefly examined. The number of diffraction-quality crystals and the time of appearance of crystals increased in the order HTS-80, Crystal Finder, TERA. With the HTS-80 and Crystal Finder, the time of appearance was short and the rate of salt crystallization was low. With the TERA, the number of diffraction-quality crystals was high, while the time of appearance was long and the rate of salt crystallization was relatively high. For the protein samples exhibiting low crystallization success rates, there were few crystallization conditions that were common to the robots used. In some cases, the success rate depended greatly on the robot used. The TOPAZ showed the shortest time of appearance and the highest success rate, although the crystals obtained were too small for diffraction studies. These results showed that the combined use of different robots significantly increases the chance of obtaining crystals, especially for proteins exhibiting low crystallization success rates. The structures of 360 of 944 purified proteins have been successfully determined through the combined use of an HTS-80 and a TERA.
doi:10.1107/S1744309108013572
PMCID: PMC2496871  PMID: 18540056
protein crystallization; crystallization screening; crystallization success rates
20.  Crystallization and preliminary X-ray analysis of reducing-end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125 
Reducing-end-xylose releasing exo-oligoxylanase (Rex) from B. halodurans C-125 was crystallized. A diffraction data set was collected to 1.35 Å resolution.
The reducing-end xylose-releasing exo-oligoxylanase (Rex) from Bacillus halodurans C-125, a novel family GH8 glycoside hydrolase, was crystallized by the hanging-drop vapour-diffusion method using 13.6 mg ml−1 purified Rex, 5.6%(v/v) polyethylene glycol 4000, 70 mM sodium acetate pH 4.6 and 30%(v/v) glycerol. Suitable crystals grew after incubation for 5 d at 293 K. The crystals belonged to space group P212121, with unit-cell parameters a = 52.69, b = 86.02, c = 87.92 Å. X-ray diffraction data were collected at a resolution of 1.35 Å.
doi:10.1107/S1744309105003635
PMCID: PMC1952294  PMID: 16511021
reducing-end xylose-releasing exo-oligoxylanase; glycoside hydrolases
21.  Crystallization and X-ray diffraction analysis of human CLEC-2 
Recombinant human CLEC-2 was crystallized in the orthorhombic space group P212121 and X-ray diffraction data were collected to 2.0 Å.
The human C-type lectin-like protein CLEC-2 has recently been shown to be expressed on the surface of platelets and to function as a receptor for the snake-venom protein rhodocytin. The C-type lectin-like domain (CTLD) of CLEC-2 was expressed in Escherichia coli, refolded and purified. Crystals of this recombinant CLEC-2 were grown by sitting-drop vapour diffusion using polyethylene glycol (PEG) 6000 as a precipitant. After optimization, crystals were grown which diffracted to 2.0 Å using in-house radiation (λ = 1.5418 Å). These crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 35.407, b = 55.143, c = 56.078 Å. The presence of one molecule per asymmetric unit is consistent with a crystal volume per unit weight (V M) of 1.82 Å3 Da−1 and a solvent content of 32.6%. These results suggest that crystals producing diffraction of this quality will be suitable for the structural determination of human CLEC-2.
doi:10.1107/S1744309105037991
PMCID: PMC1978148  PMID: 16511244
CLEC-2; CLEC1B; rhodocytin; aggretin; C-type lectins; platelets; thrombosis
22.  Crystallization and preliminary X-ray crystallographic analysis of a galactose-specific lectin from Dolichos lablab  
The galactose-specific lectin from the seeds of a leguminous plant, D. lablab, has been crystallized. Molecular-replacement solution using 3.0 Å X-ray diffraction data showed the lectin to be a tetramer.
The galactose-specific lectin from the seeds of Dolichos lablab has been crystallized using the hanging-drop vapour-diffusion technique. The crystals belong to space group P1, with unit-cell parameters a = 73.99, b = 84.13, c = 93.15 Å, α = 89.92, β = 76.01, γ = 76.99°. X-ray diffraction data to a resolution of 3.0 Å have been collected under cryoconditions (100 K) using a MAR imaging-plate detector system mounted on a rotating-anode X-ray generator. Molecular-replacement calculations carried out using the available structures of legume lectins as search models revealed that the galactose-specific lectin from D. lablab forms a tetramer similar to soybean agglutinin; two such tetramers are present in the asymmetric unit.
doi:10.1107/S1744309106001448
PMCID: PMC2150945  PMID: 16511291
Dolichos lablab; galactose-specific lectins; legume lectins
23.  Purification, crystallization and preliminary X-ray crystallographic analysis of rice lectin from Oryza sativa  
Rice lectin was crystallized and analyzed by X-ray crystallography.
Lectins with sugar-binding specificity are widely distributed in higher plants and various other species. The expression of rice lectin from Oryza sativa is up-regulated in the growing coleoptile when anaerobic stress persists. A rice lectin of molecular weight 15.2 kDa has been crystallized using the hanging-drop vapour-diffusion method. From the diffraction of the lectin crystals at 1.93 Å resolution, the unit cell belongs to space group P31, with unit-cell parameters a = 98.58, b = 98.58, c = 44.72 Å. Preliminary analysis indicates that there are two lectin molecules in an asymmetric unit with a large solvent content, 70.1%.
doi:10.1107/S1744309105040698
PMCID: PMC2150942  PMID: 16511272
lectins; rice
24.  Crystallization and preliminary X-ray crystallographic analysis of the α-2,6-sialyltransferase PM0188 from Pasteurella multosida  
The α-2,6-sialyltransferase PM0188 from P. multocida was purified using affinity-column chromatographic methods and crystallized using the hanging-drop vapour-diffusion method at 293 K.
Sialyltransferase is an enzyme that transfers the sialic acid moiety from cytidine-5-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the carbohydrate group of various glycoproteins. These glycoproteins are involved in inflammation, embryogenesis, immune defence and metastasis of cancer cells by cell–cell interactions or cell–matrix interactions. The α-2,6-sialyltransferase PM0188 from Pasteurella multocida was purified using affinity-column chromatographic methods and crystallized using the hanging-drop vapour-diffusion method at 293 K. MAD data were collected to 1.9 Å resolution from an SeMet-substituted crystal. The crystal belongs to space group P21, with unit-cell parameters a = 52.9, b = 61.0, c = 64.6 Å, α = γ = 90, β = 112.3°. Assuming the presence of one molecule in the asymmetric unit, the solvent content is estimated to be about 45%.
doi:10.1107/S1744309106000844
PMCID: PMC2150949  PMID: 16511286
PM0188; sialyltransferases
25.  Crystallization and initial X-ray diffraction studies of scaffolding protein (gp7) of bacteriophage ϕ29 
ϕ29 bacteriophage scaffolding protein (gp7) has been overproduced in E. coli, purified, crystallized and characterized by X-ray diffraction. Two distinct crystal forms were obtained and a diffraction data set was collected to 1.8 Å resolution.
The Bacillus subtilis bacteriophage ϕ29 scaffolding protein (gp7) has been crystallized by the hanging-drop vapour-diffusion method at 293 K. Two new distinct crystal forms that both differed from a previously crystallized and solved scaffolding protein were grown under the same conditions. Form I belongs to the primitive tetragonal space group P41212, with unit-cell parameters a = b = 77.13, c = 37.12 Å. Form II crystals exhibit an orthorhombic crystal form, with space group C222 and unit-cell parameters a = 107.50, b = 107. 80, c = 37.34 Å. Complete data sets have been collected to 1.78 and 1.80 Å for forms I and II, respectively, at 100 K using Cu Kα X-rays from a rotating-anode generator. Calculation of a V M value of 2.46 Å3 Da−1 for form I suggests the presence of one molecule in the asymmetric unit, corresponding to a solvent content of 50.90%, whereas form II has a V M of 4.80 Å3 Da−1 with a solvent content of 48.76% and two molecules in the asymmetric unit. The structures of both crystal forms are being determined by the molecular-replacement method using the coordinates of the published crystal structure of gp7.
doi:10.1107/S1744309105008511
PMCID: PMC1952437  PMID: 16511059
scaffolding protein; bacteriophage ϕ29

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