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1.  Expression, crystallization and preliminary X-ray crystallographic analysis of XometC, a cystathionine γ-lyase-like protein from Xanthomonas oryzae pv. oryzae  
XometC, a cystathionine γ-lyase-like protein from X. oryzae pv. oryzae and an antibacterial drug-target protein against bacterial blight, was cloned, purified and crystallized. Preliminary X-ray crystallographic analysis of XometC crystals was carried out.
Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight of rice (Oryza sativa L.), one of the most devastating diseases of rice in most rice-growing countries. XometC, a cystathionine γ-lyase (CGL) like protein that is an antibacterial drug-target protein against Xoo, was cloned, expressed, purified and crystallized. CGL catalyzes the second step in the reverse-transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids cysteine and methionine. Crystals of two different shapes, plate-shaped and pyramid-shaped, diffracted to 2.9 and 3.2 Å resolution and belonged to the primitive orthogonal space group P212121 and the tetragonal space group P41 (or P43), with unit-cell parameters a = 73.0, b = 144.9, c = 152.3 Å and a = b = 78.2, c = 300.7 Å, respectively. For the P212121 crystals, three or four monomers exist in the asymmetric unit with a corresponding V M of 3.02 or 2.26 Å3 Da−1 and a solvent content of 59.3 or 45.7%. For the P41 (or P43) crystals, four or five monomers exist in the asymmetric unit with a corresponding V M of 2.59 or 2.09 Å3 Da−1 and a solvent content of 52.5 or 40.6%.
doi:10.1107/S1744309108021155
PMCID: PMC2494973  PMID: 18678949
bacterial blight; cystathionine γ-lyase; reverse transsulfuration pathway; Xanthomonas oryzae pv. oryzae
2.  Crystallization and preliminary crystallographic studies of LipA, a secretory lipase/esterase from Xanthomonas oryzae pv. oryzae  
The crystallization and preliminary crystallographic studies of LipA, a lipase/esterase secreted by X. oryzae pv. oryzae during its infection of rice plants, are reported.
Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. Several enzymes that are secreted through the type II secretion system of this bacterium play an important role in the plant–microbe interaction, being important for virulence and also being able to induce potent host defence responses. One of these enzymes is a secretory lipase/esterase, LipA, which shows a very weak homology to other bacterial lipases and gives a positive tributyrin plate assay. In this study, LipA was purified from the culture supernatant of an overexpressing clone of X. oryzae pv. oryzae and two types of crystals belonging to space group C2 but with two different unit-cell parameters were obtained using the hanging-drop vapour-diffusion method. Type I crystals diffract to a maximum resolution of 1.89 Å and have unit-cell parameters a = 93.1, b = 62.3, c = 66.1 Å, β = 90.8°. Type II crystals have unit-cell parameters a = 103.6, b = 54.6, c = 66.3 Å, β = 92.6° and diffract to 1.86 Å. Solvent-content analysis shows one monomer in the asymmetric unit in both the crystal forms.
doi:10.1107/S1744309107034367
PMCID: PMC2335158  PMID: 17671374
secretory lipases; Xanthomonas oryzae pv. oryzae; plant pathogens
3.  Purification, crystallization and preliminary X-ray crystallographic analysis of rice Bowman–Birk inhibitor from Oryza sativa  
Rice Bowman–Birk inhibitor was expressed and crystallized.
Bowman–Birk inhibitors (BBIs) are cysteine-rich proteins with inhibitory activity against proteases that are widely distributed in monocot and dicot species. The expression of rice BBI from Oryza sativa is up-regulated and induced by pathogens or insects during germination of rice seeds. The rice BBI (RBTI) of molecular weight 15 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of rice BBI crystals at a resolution of 2.07 Å, the unit cell belongs to space group P212121, with unit-cell parameters a = 74.37, b = 96.69, c = 100.36 Å. Preliminary analysis indicates four BBI molecules in an asymmetric unit, with a solvent content of 58.29%.
doi:10.1107/S1744309106014795
PMCID: PMC2243081  PMID: 16754971
Bowman–Birk inhibitors; rice
4.  Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of the co-chaperonin XoGroES from Xanthomonas oryzae pv. oryzae  
Bacterial blight, an infectious disease caused by X. oryzae pv. oryzae (Xoo), causes huge rice-production losses in most rice-cultivating countries. The co-chaperonin of Xoo, XoGroES, an important protein for protein folding, was cloned from Xoo, purified and crystallized for atomic resolution structure determination.
Bacterial blight (BB), a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo), causes serious production losses of rice in Asian countries. Protein misfolding may interfere with the function of proteins in all living cells and must be prevented to avoid cellular disaster. All cells naturally contain molecular chaperones that assist the unfolded proteins in folding into the native structure. One of the well characterized chaperone complexes is GroEL–GroES. GroEL, which consists of two chambers, captures misfolded proteins and refolds them. GroES is a co-chaperonin protein that assists the GroEL protein as a lid that temporarily closes the chamber during the folding process. Xoo4289, the GroES gene from Xoo, was cloned and expressed for X-ray crystallographic study. The purified protein (XoGroES) was crystallized using the hanging-drop vapour-diffusion method and a crystal diffracted to 2.0 Å resolution. The crystal belonged to the hexagonal space group P61, with unit-cell parameters a = 64.4, c = 36.5 Å. The crystal contains a single molecule in the asymmetric unit, with a corresponding V M of 2.05 Å3 Da−1 and a solvent content of 39.9%.
doi:10.1107/S1744309110038820
PMCID: PMC3079969  PMID: 21206021
Xanthomonas oryzae pv. oryzae; bacterial blight; co-chaperonins; Xoo4289
5.  Crystallization and preliminary X-ray crystallographic studies of dehydroascorbate reductase (DHAR) from Oryza sativa L. japonica  
A dehydroascorbate reductase (DHAR) from rice (O. sativa L. japonica) was overexpressed, purified and crystallized. The crystals belonged to space group P21 and a complete native data set was obtained to a resolution of 1.9 Å.
Dehydroascorbate reductase from Oryza sativa L. japonica (OsDHAR), a key enzyme in the regeneration of vitamin C, maintains reduced pools of ascorbic acid to detoxify reactive oxygen species. In previous studies, the overexpression of OsDHAR in transgenic rice increased grain yield and biomass as well as the amount of ascorbate, suggesting that ascorbate levels are directly associated with crop production in rice. Hence, it has been speculated that the increased level of antioxidants generated by OsDHAR protects rice from oxidative damage and increases the yield of rice grains. However, the crystal structure and detailed mechanisms of this important enzyme need to be further elucidated. In this study, recombinant OsDHAR protein was purified and crystallized using the sitting-drop vapour-diffusion method at pH 8.0 and 298 K. Plate-shaped crystals were obtained using 0.15 M potassium bromide, 30%(w/v) PEG MME 2000 as a precipitant, and the crystals diffracted to a resolution of 1.9 Å on beamline 5C at the Pohang Accelerator Laboratory. The X-ray diffraction data indicated that the crystal contained one OsDHAR molecule in the asymmetric unit and belonged to space group P21 with unit-cell parameters a = 47.03, b = 48.38, c = 51.83 Å, β = 107.41°.
doi:10.1107/S2053230X14009133
PMCID: PMC4051537  PMID: 24915093
ascorbate; dehydroascorbate reductase; Oryza sativa L. japonica
6.  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
7.  Crystallization and X-ray analysis of the salmon-egg lectin SEL24K 
The 24 kDa egg lectin of Chinook salmon (Oncorhynchus tshawytscha) was purified by affinity chromatography from salmon eggs and crystallized by the hanging-drop vapor-diffusion method using 15/4 EO/OH (pentaerythritol ethoxylate) as a precipitant.
The 24 kDa egg lectin of Chinook salmon (Oncorhynchus tshawytscha) is released from the egg during the cortical reaction. The lectin functions in blocking polyspermy during the fertilization process. The egg lectin was purified by affinity chromatography from salmon eggs and crystallized by the hanging-drop vapor-diffusion method using 15/4 EO/OH (pentaerythritol ethoxylate) as a precipitant. The crystal diffracted synchrotron-radiation X-rays to 1.63 Å resolution. The crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 93.0, b = 73.6, c = 113.6 Å, α = 90, β = 92.82, γ = 90°. The crystal is likely to contain eight molecules in the asymmetric unit (V M = 2.3 Å3 Da−1), corresponding to a solvent content of 45.5%. A self-rotation function suggests an arrangement with 222 point symmetry within the asymmetric unit.
doi:10.1107/S1744309107015345
PMCID: PMC2335001  PMID: 17565179
lectins; salmon; polyspermy
8.  Crystallization and preliminary X-ray crystallographic analysis of the XoGroEL chaperonin from Xanthomonas oryzae pv. oryzae  
The XoGroEL gene (XOO_4288) was cloned from X. oryzae pv. oryzae and the protein was expressed, purified and crystallized. A preliminary X-ray crystallographic analysis of an XoGroEL crystal was performed.
Along with the co-chaperonin GroES, the chaperonin GroEL plays an essential role in enhancing protein folding or refolding and in protecting proteins against misfolding and aggregation in the cellular environment. The XoGroEL gene (XOO_4288) from Xanthomonas oryzae pv. oryzae was cloned and the protein was expressed, purified and crystallized. The purified XoGroEL protein was crystallized using the hanging-drop vapour-diffusion method and a crystal diffracted to a resolution of 3.4 Å. The crystal belonged to the orthorhombic space group P212121 with 14 monomers in the asymmetric unit, with a corresponding V M of 2.7 Å3 Da−1 and a solvent content of 54.5%.
doi:10.1107/S2053230X14006591
PMCID: PMC4014328  PMID: 24817719
GroEL; chaperone; Xanthomonas oryzae pv. oryzae (Xoo)
9.  Crystallization and preliminary X-ray diffraction analysis of the lectin from Canavalia boliviana Piper seeds 
Canavalia boliviana lectin (Cbol) was purified using a Sephadex G-50 column and crystallized in the presence of X-Man by hanging-drop vapour diffusion at 293 K. After optimization, crystals suitable for diffraction were obtained using 0.1 M HEPES pH 7.5 and 3.0 M sodium formate.
Plant lectins are the most studied group of carbohydrate-binding proteins. Despite the high similarity between the members of the Diocleinae subtribe (Leguminosae) group, they present differing biological activities. Canavalia boliviana lectin (Cbol) was purified using a Sephadex G-50 column and crystallized in the presence of X-Man by hanging-drop vapour diffusion at 293 K. After optimization, crystals suitable for diffraction were obtained under the condition 0.1 M HEPES pH 7.5 and 3.0 M sodium formate. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 126.70, b = 66.64, c = 64.99 Å, α = 90.0, β = 120.8, γ = 90.0°. Assuming the presence of a dimer in the asymmetric unit, the solvent content was estimated to be about 46%. A complete data set was collected at 1.5 Å resolution.
doi:10.1107/S1744309109000797
PMCID: PMC2650465  PMID: 19255467
lectins; Canavalia boliviana Piper
10.  Expression, crystallization and preliminary X-ray crystallographic analysis of Xoo2316, a predicted 6-­phosphogluconolactonase, from Xanthomonas oryzae pv. oryzae  
Xoo2316 from X. oryzae pv. oryzae, a predicted 6-phosphogluconolactonase, the second enzyme of the pentose phosphate pathway, has been cloned, purified and crystallized. A native data set was collected to 2.4 Å resolution and a MAD data set was collected to 2.5 Å resolution.
Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, which is one of the most devastating diseases of rice (Oryza sativa L.) in many rice-growing countries. The coding sequence of Xoo2316 (a predicted 6-­phosphogluconolactonase; 6PGL) from Xoo was cloned and expressed in Escherichia coli. 6PGL is an enzyme that is involved in the second step of the pentose phosphate pathway, which is essential for the synthesis of nucleotide sugars and NADPH, the main source of reducing power. The protein was purified and crystallized in order to elucidate the molecular basis for its enzymatic reaction. Native crystals diffracted to 2.4 Å resolution and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 40.0, b = 65.1, c = 78.8 Å. A monomer exists in the asymmetric unit with a corresponding V M of 1.93 Å3 Da−1 and a solvent content of 36.5%.
doi:10.1107/S1744309108025852
PMCID: PMC2581699  PMID: 18997330
bacterial blight; 6-phosphogluconolactonase; PIP box; pentose phosphate pathway; Xanthomonas oryzae pv. oryzae
11.  Purification, crystallization and preliminary X-ray analysis of OsAREB8 from rice, a member of the AREB/ABF family of bZIP transcription factors, in complex with its cognate DNA 
OsAREB8 from rice (O. sativa), a member of the AREB/ABF family of bZIP transcription factors, was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. A crystal of OsAREB8 in complex with its cognate DNA diffracted X-rays to 3.65 Å resolution.
The AREB/ABF family of bZIP transcription factors play a key role in drought stress response and tolerance during the vegetative stage in plants. To reveal the DNA-recognition mechanism of the AREB/ABF family of proteins, the bZIP domain of OsAREB8, an AREB/ABF-family protein from Oryza sativa, was expressed in Escherichia coli, purified and crystallized with its cognate DNA. Crystals of the OsAREB8–DNA complex were obtained by the sitting-drop vapour-diffusion method at 277 K with a reservoir solution consisting of 50 mM MES pH 6.4, 29% MPD, 2 mM spermidine, 20 mM magnesium acetate and 100 mM sodium chloride. A crystal diffracted X-rays to 3.65 Å resolution and belonged to space group C222, with unit-cell parameters a = 155.1, b = 206.7, c = 38.5 Å. The crystal contained one OsAREB8–DNA complex in the asymmetric unit.
doi:10.1107/S1744309112009384
PMCID: PMC3325828  PMID: 22505428
AREB/ABF family; stress response; Oryza sativa
12.  Purification, crystallization and preliminary X-ray crystallographic analysis of rice bifunctional α-amylase/subtilisin inhibitor from Oryza sativa  
The crystallization of rice α-amylase/subtilisin bifunctional inhibitor is reported.
Rice bifunctional α-amylase/subtilisin inhibitor (RASI) can inhibit both α-­amylase from larvae of the red flour beetle (Tribolium castaneum) and subtilisin from Bacillus subtilis. The synthesis of RASI is up-regulated during the late milky stage in developing seeds. The 8.9 kDa molecular-weight RASI from rice has been crystallized using the hanging-drop vapour-diffusion method. According to 1.81 Å resolution X-ray diffraction data from rice RASI crystals, the crystal belongs to space group P21212, with unit-cell parameters a = 79.99, b = 62.95, c = 66.70 Å. Preliminary analysis indicates two RASI molecules in an asymmetric unit with a solvent content of 44%.
doi:10.1107/S1744309106023335
PMCID: PMC2242909  PMID: 16880545
α-amylase/subtilisin inhibitor; rice
13.  Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of 3-dehydroquinate synthase, Xoo1243, from Xanthomonas oryzae pv. oryzae  
Bacterial blight is a destructive disease of rice that is caused by X. oryzae pv. oryzae (Xoo). Dehydroquinate synthase, which is the second enzyme of the shikimate pathway, was cloned from Xoo1243 (aroB), purified and crystallized in order to elucidate its three-dimensional structure.
The disease bacterial blight results in serious production losses of rice in Asian countries. The aroB gene encoding dehydroquinate synthase (DHQS), which is a potential antibiotic target, was identified from the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo). DHQS plays an essential role in the synthesis of aromatic compounds in the shikimate pathway. The aroB gene (Xoo1243) was cloned from Xoo and the corresponding DHQS protein was subsequently overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion method and yielded crystals that diffracted to 2.5 Å resolution. The crystals belonged to the tetragonal space group P43212, with unit-cell parameters a = b = 118.2, c = 98.2 Å. According to a Matthews coefficient calculation, the crystal contained two molecules in the asymmetric unit, with a corresponding V M of 2.06 Å3 Da−1 and a solvent content of 40.4%.
doi:10.1107/S1744309108033575
PMCID: PMC2593707  PMID: 19052366
aroB; bacterial blight; 3-dehydroquinate synthase; shikimate pathway; Xanthomonas oryzae pv. oryzae
14.  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
15.  Crystallization and preliminary X-ray studies of a galactose-specific lectin from the seeds of Spatholobus parviflorus  
A galactose specific lectin was purified from the seeds of a tropical plant, Spatholobus parviflorus. Its X-ray crystallographic structure was solved by the molecular replacement method.
A galactose-specific seed lectin was purified from the legume Spatholobus parviflorus and crystallized using the hanging-drop vapour-diffusion technique. The crystals belonged to space group P1, with unit-cell parameters a = 60.998, b = 60.792, c = 78.179 Å, α = 101.32, β = 91.38, γ = 104.32°. X-ray diffraction data were collected under cryoconditions (100 K) to a resolution of 2.04 Å using a MAR image-plate detector system mounted on a rotating-anode X-ray (Cu Kα) generator. Molecular replacement using legume-lectin coordinates as a search model gave a tetrameric structure.
doi:10.1107/S174430911101387X
PMCID: PMC3107147  PMID: 21636916
galactose-specific lectins; Spatholobus parviflorus; seed lectins
16.  Expression, crystallization and preliminary X-ray crystallographic analysis of Xoo0352, d-alanine-d-alanine ligase A, from Xanthomonas oryzae pv. oryzae  
Xoo0352, which encodes d-alanine-d-alanine ligase A (DdlA), from X. oryzae pv. oryzae was cloned, purified and crystallized. Preliminary X-ray crystallographic analysis of DdlA crystals was performed.
Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB), which is one of the most devastating diseases of rice in most rice-growing countries. d-­Alanine-d-alanine ligase A (DdlA), coded by the Xoo0352 gene, was expressed, purified and crystallized. DdlA is an enzyme that is involved in d-­alanine metabolism and the biosynthesis of an essential bacterial peptidoglycan precursor, in which it catalyzes the formation of d-alanyl-d-alanine from two d-­alanines, and is thus an attractive antibacterial drug target against Xoo. The DdlA crystals diffracted to 2.3 Å resolution and belonged to the primitive tetragonal space group P43212, with unit-cell parameters a = b = 83.0, c = 97.6 Å. There is one molecule in the asymmetric unit, with a corresponding V M of 1.88 Å3 Da−1 and a solvent content of 34.6%. The initial structure was determined by molecular replacement using d-alanine-d-alanine ligase from Staphylococcus aureus (PDB code 2i87) as a template model.
doi:10.1107/S1744309108028820
PMCID: PMC2593706  PMID: 19052362
bacterial blight; d-alanine-d-alanine ligase; peptidoglycan biosynthesis; Xanthomonas oryzae pv. oryzae
17.  Purification, crystallization and preliminary X-ray crystallographic analysis of glycosyltransferase-1 from Bacillus cereus  
The purification, crystallization and preliminary X-ray crystallographic analysis of B. cereus glycosyltransferase-1 are reported.
Glycosyltransferases (GTs), which are distributed widely in various organisms, including bacteria, fungi, plants and animals, play a role in synthesizing biological compounds. Glycosyltransferase-1 from Bacillus cereus (BcGT-1), which is capable of transferring glucose to small molecules such as kaempferol and quercetin, has been identified as a member of the family 1 glycosyltransferases which utilize uridine diphosphate glucose (UDP-glucose) as the sugar donor. BcGT-1 (molecular mass 45.5 kDa) has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction of BcGT-1 crystals to 2.10 Å resolution, the crystal belonged to space group P1, with unit-cell parameters a = 54.56, b = 84.81, c = 100.12 Å, α = 78.36, β = 84.66, γ = 84.84°. Preliminary analysis indicates the presence of four BcGT-1 molecules in the asymmetric unit with a solvent content of 50.27%.
doi:10.1107/S2053230X14014629
PMCID: PMC4157424  PMID: 25195897
glycosyltransferase; Bacillus cereus
18.  Purification, characterization and preliminary X-ray crystallographic studies of monodehydroascorbate reductase from Oryza sativa L. japonica  
Monodehydroascorbate reductase (OsMDHAR) from O. sativa L. japonica was expressed as the active form using E. coli strain NiCo21 (DE3). X-ray diffraction data were collected to 1.9 Å resolution.
Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is a key enzyme in the reactive oxygen species (ROS) detoxification system of plants. The participation of MDHAR in ascorbate (AsA) recycling in the ascorbate–glutathione cycle is important in the acquired tolerance of crop plants to abiotic environmental stresses. Thus, MDHAR represents a strategic target protein for the improvement of crop yields. Although physiological studies have intensively characterized MDHAR, a structure-based functional analysis is not available. Here, a cytosolic MDHAR (OsMDHAR) derived from Oryza sativa L. japonica was expressed using Escherichia coli strain NiCo21 (DE3) and purified. The purified OsMDHAR showed specific enzyme activity (approximately 380 U per milligram of protein) and was crystallized using the hanging-drop vapour-diffusion method at pH 8.0 and 298 K. The crystal diffracted to 1.9 Å resolution and contained one molecule in the asymmetric unit (the Matthews coefficient V M is 1.98 Å3 Da−1, corresponding to a solvent content of 38.06%) in space group P41212 with unit-cell parameters a = b = 81.89, c = 120.4 Å. The phase of the OsMDHAR structure was resolved by the molecular-replacement method using a ferredoxin reductase from Acidovorax sp. strain KKS102 (PDB entry 4h4q) as a model.
doi:10.1107/S2053230X14015908
PMCID: PMC4157428  PMID: 25195901
monodehydroascorbate reductase; Oryza sativa L. japonica; abiotic stress; ascorbate regeneration
19.  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
20.  Crystallization and preliminary X-ray diffraction analysis of a galactose-specific lectin from the seeds of Butea monosperma  
A galactose specific lectin was purified from the seeds of a tropical tree, Butea monosperma. Its X-ray structure was solved by molecular replacement.
The galactose-specific lectin from the seeds of Butea monosperma has been crystallized by the hanging-drop vapour-diffusion technique. The crystals belonged to space group P1, with unit-cell parameters a = 78.45, b = 78.91, c = 101.85 Å, α = 74.30, β = 76.65, γ = 86.88°. X-ray diffraction data were collected to a resolution of 2.44 Å under cryoconditions (100 K) using a MAR image-plate detector system mounted on a rotating-anode X-ray generator. Molecular-replacement calculations carried out using the coordinates of several structures of legume lectins as search models indicate that the galactose-specific lectin from B. monosperma forms an octamer.
doi:10.1107/S1744309111006853
PMCID: PMC3080167  PMID: 21505258
galactose-specific lectin; Butea monosperma
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.  Purification, crystallization and preliminary X-ray analysis of rice BGlu1 β-glucosidase with and without 2-deoxy-2-fluoro-β-d-glucoside 
Rice BGlu1 β-glucosidase was purified from recombinant E. coli and crystallized with and without the inhibitor 2-deoxy-2-fluoro-β-d-glucose. The crystals diffracted to 2.15 and 2.75 Å, respectively.
Rice (Oryza sativa) BGlu1 β-glucosidase was expressed in Escherichia coli with N-terminal thioredoxin and hexahistidine tags and purified by immobilized metal-affinity chromatography (IMAC). After removal of the N-terminal tags, cation-exchange and S-200 gel-filtration chromatography yielded a 50 kDa BGlu1 with >95% purity. The free enzyme and a complex with 2,4-dinitrophenyl-2-­deoxy-2-fluoro-β-d-glucopyranoside inhibitor were crystallized by microbatch and hanging-drop vapour diffusion. Small tetragonal crystals of BGlu1 with and without inhibitor grew in 18%(w/v) PEG 8000 with 0.1 M sodium cacodylate pH 6.5 and 0.2 M zinc acetate. Crystals of BGlu1 with inhibitor were streak-seeded into 23%(w/v) PEG MME 5000, 0.2 M ammonium sulfate, 0.1 M MES pH 6.7 to yield larger crystals. Crystals with and without inhibitor diffracted to 2.15 and 2.75 Å resolution, respectively, and had isomorphous orthorhombic unit cells belonging to space group P212121.
doi:10.1107/S1744309106027084
PMCID: PMC2242908  PMID: 16880561
BGlu1 β-glucosidase; 2,4-dinitrophenyl-2-­deoxy-2-fluoro-β-d-glucopyranoside; rice
23.  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
24.  Crystallization and molecular-replacement studies of the monoclonal antibody mAbR310 specific for the (R)-HNE-modified protein 
Antigen-free Fab fragment of mAbR310, which recognizes (R)-HNE modified protein, has been crystallized. Initial phases have been obtained by molecular replacement.
4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE–histidine Michael addition-type adduct possessing three chiral centres in the cyclic hemiacetal structure. Monoclonal antibodies against HNE-modified protein have been widely used for assessing oxidative stress in vitro and in vivo. Here, the purification, crystallization and preliminary crystallographic analysis of a Fab fragment of novel monoclonal antibody R310 (mAbR310), which recognizes (R)-HNE-modified protein, are reported. The Fab fragment of mAbR310 was obtained by digestion with papain, purified and crystallized. Using hanging-drop vapour-diffusion crystallization techniques, crystals of mAbR310 Fab were obtained. The crystal belongs to the monoclinic space group C2 (unit-cell parameters a = 127.04, b = 65.31, c = 64.29 Å, β = 118.88°) and diffracted X-rays to a resolution of 1.84 Å. The asymmetric unit contains one molecule of mAbR310, with a corresponding crystal volume per protein weight of 2.51 Å3 Da−1 and a solvent content of 51.0%.
doi:10.1107/S1744309106016630
PMCID: PMC2243084  PMID: 16754982
mAbR310; monoclonal antibodies; Fab fragments
25.  Purification, crystallization and preliminary crystallographic investigation of FrnE, a disulfide oxidoreductase from Deinococcus radiodurans  
drFrnE, a disulfide oxidoreductase from D. radiodurans, was crystallized and preliminary diffraction data were obtained. The crystal belonged to space group P21221 and diffracted to 1.8 Å resolution.
In prokaryotes, Dsb proteins catalyze the formation of native disulfide bonds through an oxidative folding pathway and are part of the cell machinery that protects proteins from oxidative stress. Deinococcus radiodurans is an extremophile which shows unparalleled resistance to ionizing radiation and oxidative stress. It has a strong mechanism to protect its proteome from oxidative damage. The genome of Deinococcus shows the presence of FrnE, a Dsb protein homologue that potentially provides the bacterium with oxidative stress tolerance. Here, crystallization and preliminary X-ray crystallographic analysis of FrnE from D. radiodurans are reported. Diffraction-quality single crystals were obtained using the hanging-drop vapour-diffusion method with reservoir solution consisting of 100 mM sodium acetate pH 5.0, 10% PEG 8000, 15–20% glycerol. Diffraction data were collected on an Agilent SuperNova system using a microfocus sealed-tube X-ray source. The crystal diffracted to 1.8 Å resolution at 100 K. The space group of the crystal was found to be P21221, with unit-cell parameters a = 47.91, b = 62.94, c = 86.75 Å, α = β = γ = 90°. Based on Matthews coefficient analysis, one monomer per asymmetric unit is present in the crystal, with a solvent content of approximately 45%.
doi:10.1107/S2053230X14020330
PMCID: PMC4231861  PMID: 25372826
Deinococcus radiodurans; FrnE; disulfide oxidoreductase; oxidative stress; Dsb proteins

Results 1-25 (279753)