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1.  Crystallization and preliminary X-ray diffraction analysis of an anti-H(O) lectin from Lotus tetragonolobus seeds 
The seed lectin from Lotus tetragonolobus (LTA) has been crystallized. The best crystals grew over several days and were obtained using the vapour-diffusion method at a constant temperature of 293 K.
The seed lectin from Lotus tetragonolobus (LTA) has been crystallized. The best crystals grew over several days and were obtained using the vapour-diffusion method at a constant temperature of 293 K. A complete structural data set was collected at 2.00 Å resolution using a synchrotron-radiation source. LTA crystals were found to be monoclinic, belonging to space group P21, with unit-cell parameters a = 68.89, b = 65.83, c = 102.53 Å, α = γ = 90, β = 92°. Molecular replacement yielded a solution with a correlation coefficient and R factor of 34.4 and 51.6%, respectively. Preliminary analysis of the molecular-replacement solution indicates a new quaternary association in the LTA structure. Crystallographic refinement is under way.
doi:10.1107/S1744309106021312
PMCID: PMC2242948  PMID: 16820693
LTA; Lotus tetragonolobus
2.  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
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 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
5.  Crystallization, data collection and phasing of two digestive lysozymes from Musca domestica  
The digestive lysozymes 1 and 2 from M. domestica were crystallized by vapour diffusion. The crystallographic data were processed to a maximum resolution of 1.9 Å in both cases.
Lysozymes are mostly known for their defensive role against bacteria, but in several animals lysozymes have a digestive function. Here, the initial crystallographic characterization of two digestive lysozymes from Musca domestica are presented. The proteins were crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate or PEG/2-propanol as the precipitant. X-ray diffraction data were collected to a maximum resolution of 1.9 Å using synchrotron radiation. The lysozyme 1 and 2 crystals belong to the monoclinic space group P21 (unit-cell parameters a = 36.52, b = 79.44, c = 45.20 Å, β = 102.97°) and the orthorhombic space group P21212 (unit-cell parameters a = 73.90, b = 96.40, c = 33.27 Å), respectively. The crystal structures were solved by molecular replacement and structure refinement is in progress.
doi:10.1107/S1744309106024201
PMCID: PMC2242927  PMID: 16880547
digestive lysozymes; Musca domestica
6.  Crystallization and preliminary X-ray crystallographic analysis of a blue-light-absorbing proteorhodopsin 
A purified blue-light-absorbing proteorhodopsin D97N mutant protein (BPR_D97N) has been crystallized using the vapour-diffusion method.
Proteorhodopsins (PRs), seven-transmembrane chromoproteins with retinal as a chromophore, are light-driven proton pumps. To elucidate the light-driven proton-pumping mechanism of PRs, a pET28a vector containing the blue-light-absorbing proteorhodopsin (BPR) gene was constructed and the protein was overexpressed in Escherichia coli. The protein was purified by immobilized metal-ion affinity chromatography (IMAC). The purified BPR D97N mutant protein (BPR_D97N) was crystallized using the vapour-diffusion method. Preliminary X-ray diffraction data analysis showed that the crystal belonged to the orthorhombic space group P21212, with unit-cell parameters a = 161.6, b = 168.6, c = 64.7 Å. A complete data set was collected to 3.3 Å resolution using synchrotron radiation on beamline X06 of the Swiss Light Source (SLS). Molecular replacement was unsuccessful. To solve the structure of BPR_D97N by experimental phasing, selenomethionine-substituted protein crystals were prepared. These crystals diffracted to 3.0 Å resolution and a complete data set was collected on beamline BL17U of the Shanghai Synchrotron Radiation Facility (SSRF). Heavy-atom substructure determination and phasing by SAD clearly showed that the crystal contained five molecules in the asymmetric unit, with a V M of 3.26 Å3 Da−1 and a solvent content of 62.3%.
doi:10.1107/S1744309111043612
PMCID: PMC3310530  PMID: 22442222
proteorhodopsins; BPR
7.  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
8.  Crystallization and preliminary crystallographic study of a trypsin-resistant catalytic domain of human calcineurin 
A trypsin-resistant catalytic domain of human calcineurin α (A subunit, residues 20–347) was crystallized in space group P21212. An X-ray diffraction data set was collected to 2.87 Å resolution and the structure was solved by molecular replacement.
Calcineurin, a Ca2+/calmodulin-dependent serine/threonine protein phosphatase, plays a key role in a number of cellular pathways, including T-cell activation, and is an important molecular target of the immunosuppressive drugs cyclosporin A and FK506. To understand the structural basis underlying the activation of calcineurin by calmodulin, X-ray crystallography was employed to solve the three-dimensional structure of the free calcineurin catalytic domain (residues 20–347 of the A subunit). To accomplish this, a bacterially expressed glutathione S-­transferase (GST) fusion protein of the human calcineurin catalytic domain was first purified by GST-affinity chromatography. After limited digestion by trypsin, the catalytic domain (Cncat) was purified using anion-exchange and size-exclusion chromatography. Crystallization of Cncat was achieved by the hanging-drop vapour-diffusion method at pH 6.5 using PEG 6000 as precipitant. The diffraction results showed that the Cncat crystal belonged to the orthorhombic space group P21212, with unit-cell parameters a = 161.6, b = 87.4, c = 112.0 Å. There are four Cncat molecules in the asymmetric unit, with 49.5% solvent content. An X-ray diffraction data set was collected to 2.87 Å resolution and a clear molecular-replacement solution was obtained. The active site of Cncat is open to the solvent channels in the crystal packing.
doi:10.1107/S1744309112007890
PMCID: PMC3374516  PMID: 22691791
human calcineurin; catalytic domain
9.  Crystallization and preliminary X-ray diffraction studies of vitamin D3 hydroxylase, a novel cytochrome P450 isolated from Pseudonocardia autotrophica  
The purification, crystallization and preliminary X-ray diffraction studies of vitamin D3 hydroxylase isolated from P. autotrophica are reported.
Vitamin D3 hydroxylase (Vdh) is a novel cytochrome P450 monooxygenase isolated from the actinomycete Pseudonocardia autotrophica and consisting of 403 amino-acid residues. Vdh catalyzes the activation of vitamin D3 via sequential hydroxylation reactions: these reactions involve the conversion of vitamin D3 (cholecalciferol or VD3) to 25-hydroxyvitamin D3 [25(OH)VD3] and the subsequent conversion of 25(OH)VD3 to 1α,25-dihydroxyvitamin D3 [calciferol or 1α,25(OH)2VD3]. Overexpression of recombinant Vdh was carried out using a Rhodococcus erythropolis expression system and the protein was subsequently purified and crystallized. Two different crystal forms were obtained by the hanging-drop vapour-diffusion method at 293 K using polyethylene glycol as a precipitant. The form I crystal belonged to the trigonal space group P31, with unit-cell parameters a = b = 61.7, c = 98.8 Å. There is one Vdh molecule in the asymmetric unit, with a solvent content of 47.6%. The form II crystal was grown in the presence of 25(OH)VD3 and belonged to the orthorhombic system P212121, with unit-cell parameters a = 63.4, b = 65.6 c = 102.2 Å. There is one Vdh molecule in the asymmetric unit, with a solvent content of 46.7%. Native data sets were collected to resolutions of 1.75 and 3.05 Å for form I and form II crystals, respectively, using synchrotron radiation. The structure solution was obtained by the molecular-replacement method and model refinement is in progress for the form I crystal.
doi:10.1107/S1744309109007829
PMCID: PMC2664763  PMID: 19342783
cholecalciferol; CYP107; cytochrome P450 monooxygenase; hydroxylation; Pseudonocardia autotrophica; vitamin D3
10.  Purification, partial characterization and preliminary X-ray diffraction analysis of a mannose-specific lectin from Cymbosema roseum seeds 
A lectin from C. roseum seeds (CRL) has been purified, characterized and crystallized.
A lectin from Cymbosema roseum seeds (CRL) was purified, characterized and crystallized. The best crystals grew in a month and were obtained by the vapour-diffusion method using a precipitant solution consisting of 0.1 M Tris–HCl pH 7.8, 8%(w/v) PEG 3350 and 0.2 M proline at a constant temperature of 293 K. A data set was collected to 1.77 Å resolution at a synchrotron-radiation source. CRL crystals are orthorhombic, belonging to space group P212121. Crystallographic refinement and full amino-acid sequence determination are in progress.
doi:10.1107/S174430910600371X
PMCID: PMC2197170  PMID: 16511310
Cymbosema roseum; Diocleinae; lectins
11.  Crystallization and preliminary X-ray analysis of the Rieske-type [2Fe–2S] ferredoxin component of biphenyl dioxygenase from Pseudomonas sp. strain KKS102 
BphA3, a Rieske-type [2Fe–2S] ferredoxin, was crystallized by the hanging-drop vapour-diffusion method. A molecular-replacement calculation yielded a satisfactory solution.
BphA3, a Rieske-type [2Fe–2S] ferredoxin component of a biphenyl dioxygenase (BphA) from Pseudomonas sp. strain KKS102, was crystallized by the hanging-drop vapour-diffusion method. Two crystal forms were obtained from the same conditions. The form I crystal belongs to space group P21212, with unit-cell parameters a = 26.3, b = 144.3, c = 61.5 Å, and diffracted to 2.45 Å resolution. The form II crystal belongs to space group P212121, with unit-cell parameters a = 26.2, b = 121.3, c = 142.7 Å, and diffracted to 2.8 Å resolution. A molecular-replacement calculation using BphF as a search model yielded a satisfactory solution for both forms.
doi:10.1107/S1744309106017799
PMCID: PMC2243079  PMID: 16754990
ferredoxins; electron transfer; Rieske-type [2Fe–2S] clusters
12.  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
13.  Crystallization, data collection and phasing of the molybdate-binding protein of the phytopathogen Xanthomonas axonopodis pv. citri  
The molybdate-binding protein (ModA) from X. axonopodis pv. citri was crystallized with sodium molybdate in the presence of PEG or sulfate. The crystal diffracted to a maximum resolution of 1.7 Å and belongs to the orthorhombic space group C2221, with unit-cell parameters a = 68.15, b = 172.14, c = 112.04 Å.
Xanthomonas axonopodis pv. citri ModA protein is the ABC periplasmic binding component responsible for the capture of molybdate. The protein was crystallized with sodium molybdate using the hanging-drop vapour-diffusion method in the presence of PEG or sulfate. X-ray diffraction data were collected to a maximum resolution of 1.7 Å using synchrotron radiation. The crystal belongs to the orthorhombic space group C2221, with unit-cell parameters a = 68.15, b = 172.14, c = 112.04 Å. The crystal structure was solved by molecular-replacement methods and structure refinement is in progress.
doi:10.1107/S1744309106003812
PMCID: PMC2197186  PMID: 16511325
molybdate-binding protein (ModA); Xanthomonas axonopodis pv. citri
14.  Crystallization and preliminary X-ray crystallographic analysis of the oxysterol-binding protein Osh3 from Saccharomyces cerevisiae  
The PH domain and ORD of the oxysterol-binding protein Osh3 from S. cerevisae were crystallized and X-ray diffraction data were collected.
Oxysterol-binding protein (OSBP) related proteins (ORPs) are conserved from yeast to humans and are implicated in regulation of sterol homeostasis and in signal transduction pathways. Osh3 of Saccharomyces cerevisiae is a pleckstrin-homology (PH) domain-containing ORP member that regulates phosphoinositide metabolism at endoplasmic reticulum–plasma membrane contact sites. The N-terminal PH domain of Osh3 was purified and crystallized as a lysozyme fusion and the resulting crystal diffracted to 2.3 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 98.03, b = 91.31, c = 84.13 Å, β = 81.41°. With two molecules in the asymmetric unit, the Matthews coefficient was 3.13 Å3 Da−1. Initial attempts to solve the structure by molecular-replacement techniques using T4 lysozyme as a search model were successful. The C-terminal OSBP-related domain (OBD) of Osh3 was crystallized by the vapour-diffusion method and the resulting crystal diffracted to 1.5 Å resolution. The crystal was orthorhombic, belonging to space group P212121, with unit-cell parameters a = 41.57, b = 87.52, c = 100.58 Å. With one molecule in the asymmetric unit, the Matthews coefficient was 2.01 Å3 Da−1. Initial attempts to solve the structure by the single-wavelength anomalous dispersion technique using bromine were successful.
doi:10.1107/S1744309112042510
PMCID: PMC3509973  PMID: 23192032
oxysterol-binding protein; Osh3; Saccharomyces cerevisiae
15.  Purification and crystallization of Cor a 9, a major hazelnut allergen 
The major hazelnut allergen Cor a 9 was purified from the natural source and crystallized. Diffraction data were collected to 1.9 Å resolution using a synchrotron-radiation source.
Hazelnut (Corylus avellana) is one of the food sources that induce allergic reaction in a subpopulation of people with food allergy. The 11S legumin-like seed-storage protein from hazelnut has been identified as one of the major hazelnut allergens and named Cor a 9. In this study, Cor a 9 was extracted from hazelnut kernels using a high-salt solution and was purified by desalting out and FPLC to a highly purified state. Diffraction-quality single crystals were obtained using the hanging-drop vapour-diffusion method. Diffraction data were collected and a structure solution has been obtained by molecular-replacement calculations. Further refinement of the structure is currently in progress.
doi:10.1107/S1744309108039894
PMCID: PMC2628846  PMID: 19153454
hazelnuts; 11S seed-storage proteins; food allergies; tree-nut allergens
16.  Crystallization and preliminary X-ray diffraction analysis of a new chitin-binding protein from Parkia platycephala seeds 
Crystals of P. platycephala chintinase/lectin (PPL-2) belong to the orthorhombic space group P212121, with unit-cell parameters a = 55.19, b = 59.95, c = 76.60 Å. The preliminary cystal structure of PPL-2 was solved at a resolution of 1.73 Å by molecular replacement, presenting a correlation coefficient of 0.558 and an R factor of 0.439.
A chitin-binding protein named PPL-2 was purified from Parkia platycephala seeds and crystallized. Crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 55.19, b = 59.95, c = 76.60 Å, and grew over several days at 293 K using the hanging-drop method. Using synchrotron radiation, a complete structural data set was collected to 1.73 Å resolution. The preliminary crystal structure of PPL-2, determined by molecular replacement, presents a correlation coefficient of 0.558 and an R factor of 0.439. Crystallographic refinement is in progress.
doi:10.1107/S1744309105024462
PMCID: PMC1978108  PMID: 16511174
chitin-binding proteins; chitinases; Parkia platycephala; lectins
17.  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
18.  Purification, crystallization and preliminary X-ray diffraction analysis of the thiaminase type II from Staphylococcus aureus  
Crystals of the thiaminase type II from S. aureus are orthorhombic, belonging to space group P212121 with unit-cell parameters a = 103.5, b = 104.1, c = 109.6 Å, and diffracted to 2.6 Å resolution.
Thiaminase type II (TenA) catalyzes the deamination of aminopyrimidines, including the cleavage of thiamine to 4-amino-5-hydroxymethyl-2-methyl­pyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole in the metabolism of thiamine (vitamin B1), in Staphylococcus aureus (Sa). SaTenA was crystallized by the vapour-diffusion method and the resulting crystal diffracted to 2.6 Å resolution usng synchrotron radiation. The crystal is orthorhombic, belonging to space group P212121 with unit-cell parameters a = 103.5, b = 104.1, c = 109.6 Å. With four molecules in the asymmetric unit, the Matthews coefficient is 2.85 Å3 Da−1. Initial attempts to solve the structure by molecular-replacement techniques were successful.
doi:10.1107/S1744309110043174
PMCID: PMC3079971  PMID: 21206023
thiaminase type II; TenA; Staphylococcus aureus
19.  Cloning, purification, crystallization and preliminary X-ray analysis of the receiver domain of the histidine kinase CKI1 from Arabidopsis thaliana  
The crystallization of the receiver domain of the histidine kinase CYTOKININ-INDEPENDENT1 from A. thaliana is described. The crystals diffracted to 2.0 Å resolution.
The receiver domain (RD) of a sensor histidine kinase (HK) catalyses the transphosphorylation reaction during the action of HKs in hormonal and abiotic signalling in plants. Crystals of the recombinant RD of the Arabidopsis thaliana HK CYTOKININ-INDEPENDENT1 (CKI1RD) have been obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant and glycerol as a cryoprotectant. The crystals diffracted to approximately 2.4 Å resolution on beamline BW7B of the DORIS-III storage ring. The diffraction improved significantly after the use of a non-aqueous cryoprotectant. Crystals soaked in Paratone-N diffracted to at least 2.0 Å resolution on beamline BW7B and their mosaicity decreased more than tenfold. The crystals belonged to space group C2221, with unit-cell parameters a = 54.46, b = 99.82, c = 79.94 Å. Assuming the presence of one molecule of the protein in the asymmetric unit gives a Matthews coefficient V M of 2.33 Å3 Da−1. A molecular-replacement solution has been obtained and structure refinement is in progress.
doi:10.1107/S1744309109012032
PMCID: PMC2675589  PMID: 19407381
receiver domains; two-component systems; histidine kinases; cytokinins; phosphorelay; CKI1
20.  Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) from Staphylococcus aureus MSSA476 
The cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) from S. aureus MSSA476 is reported.
The gene product of the sas2203 ORF of Staphylococcus aureus MSSA476 encodes a 30 kDa molecular-weight protein with a high sequence resemblance (29% identity) to tetrameric inositol monophosphatase from Thermotoga maritima. The protein was cloned, expressed, purified to homogeneity and crystallized. Crystals appeared in several conditions and good diffraction-quality crystals were obtained from 0.2 M Li2SO4, 20% PEG 3350, 0.1 M HEPES pH 7.0 using the sitting-drop vapour-diffusion method. A complete diffraction data set was collected to 2.6 Å resolution using a Rigaku MicroMax-007 HF Cu Kα X-­ray generator and a Rigaku R-AXIS IV++ detector. The diffraction data were consistent with the orthorhombic space group P212121, with unit-cell parameters a = 49.98, b = 68.35, c = 143.79 Å, α = β = γ = 90°, and the crystal contained two molecules in the asymmetric unit.
doi:10.1107/S1744309111003496
PMCID: PMC3080153  PMID: 21505244
SAS2203; Staphylococcus aureus MSSA476; inositol monophosphatase family
21.  Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules 
Background
Lectins are mainly described as simple carbohydrate-binding proteins. Previous studies have tried to identify other binding sites, which possible recognize plant hormones, secondary metabolites, and isolated amino acid residues. We report the crystal structure of a lectin isolated from Canavalia gladiata seeds (CGL), describing a new binding pocket, which may be related to pathogen resistance activity in ConA-like lectins; a site where a non-protein amino-acid, α-aminobutyric acid (Abu), is bound.
Results
The overall structure of native CGL and complexed with α-methyl-mannoside and Abu have been refined at 2.3 Å and 2.31 Å resolution, respectively. Analysis of the electron density maps of the CGL structure shows clearly the presence of Abu, which was confirmed by mass spectrometry.
Conclusion
The presence of Abu in a plant lectin structure strongly indicates the ability of lectins on carrying secondary metabolites. Comparison of the amino acids composing the site with other legume lectins revealed that this site is conserved, providing an evidence of the biological relevance of this site. This new action of lectins strengthens their role in defense mechanisms in plants.
doi:10.1186/1472-6807-7-52
PMCID: PMC1955443  PMID: 17683532
22.  Crystallization and preliminary crystallographic analysis of a haloalkane dehalogenase, DbjA, from Bradyrhizobium japonicum USDA110 
A haloalkane dehalogenase, DbjA, was crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystal belongs to the orthorhombic system, space group P21212 and diffracts to 1.75 Å resolution.
Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. The haloalkane dehalogenase DbjA constitutes a novel substrate-specificity class with high catalytic activity for β-methylated haloalkanes. In order to reveal the mechanism of its substrate specificity, DbjA has been crystallized using the hanging-drop vapour-diffusion method. The best crystals were obtained using the microseeding technique with a reservoir solution consisting of 17–19.5%(w/v) PEG 4000, 0.2 M calcium acetate and 0.1 M Tris–HCl pH 7.7–8.0. The space group of the DbjA crystal is P21212, with unit-cell parameters a = 212.9, b = 117.8, c = 55.8 Å. The crystal diffracts to 1.75 Å resolution.
doi:10.1107/S1744309107008652
PMCID: PMC2330215  PMID: 17401198
haloalkane dehalogenases; biodegradation; α/β hydrolases; rhizobia
23.  Expression, purification, crystallization and preliminary X-ray crystallographic studies of the trehalulose synthase MutB from Pseudomonas mesoacidophila MX-45 
The trehalulose synthase MutB from P. mesoacidophila MX-45 has been crystallized in two different crystal forms and diffraction data have been collected to 1.6 and 1.8 Å, respectively.
The trehalulose synthase (MutB) from Pseudomonas mesoacidophila MX-45, belonging to glycoside hydrolase family 13, catalyses the isomerization of sucrose to trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose) and isomaltulose (α-­d-glucosylpyranosyl-1,6-d-fructofuranose) as main products and glucose and fructose in residual amounts from the hydrolytic reaction. To date, a three-dimensional structure of a sucrose isomerase that produces mainly trehalulose, as is the case for MutB, has been lacking. Crystallographic studies of this 64 kDa enzyme have therefore been initiated in order to contribute to the understanding of the molecular basis of sucrose decomposition, isomerization and of the selectivity of this enzyme that leads to the formation of different products. The MutB protein has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Two different crystal forms have been obtained: one diffracts X-rays to 1.6 Å resolution using synchrotron radiation and belongs to space group P1, with unit-cell parameters a = 63.8, b = 72.0, c = 82.2 Å, α = 67.5, β = 73.1, γ = 70.8°, while the other form diffracts to 1.8 Å resolution using synchrotron radiation and belongs to space group P21, with unit-cell parameters a = 63.7, b = 85.9, c = 119.7 Å, β = 97.7°. A molecular-replacement solution has been found using the structure of the isomaltulose synthase (PalI) from Klebsiella sp. LX3 as a search model.
doi:10.1107/S1744309104030623
PMCID: PMC1952383  PMID: 16508103
isomerases; hydrolases; trehalulose synthase
24.  Crystallization and preliminary crystallographic analysis of maganese(II)-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase from Bacillus sp. JF8 
A thermostable manganese(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase derived from Bacillus sp. JF8 was crystallized in two forms using the sitting-drop vapour-diffusion method. Both crystals diffracted to approximately 1.3 Å resolution.
A thermostable manganese(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase derived from Bacillus sp. JF8 was crystallized. The initial screening for crystallization was performed by the sitting-drop vapour-diffusion method using a crystallization robot, resulting in the growth of two crystal forms. The first crystal belonged to space group P1, with unit-cell parameters a = 62.7, b = 71.4, c = 93.6 Å, α = 71.2, β = 81.0, γ = 64.0°, and diffracted to 1.3 Å resolution. The second crystal belonged to space group I222, with unit-cell parameters a = 74.2, b = 90.8, c = 104.3 Å, and diffracted to 1.3 Å resolution. Molecular-replacement trials using homoprotocatechuate 2,3-dioxygenase from Arthrobacter globiformis (28% amino-acid sequence identity) as a search model provided a satisfactory solution for both crystal forms.
doi:10.1107/S1744309109054396
PMCID: PMC2833037  PMID: 20208161
extradiol dioxygenases; crystallization robots; metalloproteins
25.  Crystallization and preliminary X-ray diffraction analysis of Q4DV70 from Trypanosoma cruzi, a hypothetical protein with a putative thioredoxin domain 
The gene coding for Q4DV70 has been cloned and the protein overexpressed in Escherichia coli with an N-­terminal His tag. Purification of Q4DV70 was carried out by affinity and size-exclusion chromatography and the His tag was removed by TEV protease digestion. Crystals of Q4DV70 were grown using the sitting-drop vapour-diffusion method.
Q4DV70 is annotated in the Trypanosoma cruzi CL Brener genome as a hypothetical protein with a predicted thioredoxin-like fold, although the catalytic cysteine residues that are conserved in typical oxidoreductases are replaced by serine residues. Gene-expression analysis indicates that this protein is differentially expressed during the T. cruzi life cycle, suggesting that it plays an important role during T. cruzi development. The gene coding for Q4DV70 was cloned and the protein was overexpressed in Escherichia coli with an N-­terminal His tag. Purification of Q4DV70 was carried out by affinity and size-exclusion chromatography and the His tag was removed by TEV protease digestion. Crystals of Q4DV70 were grown using the sitting-drop vapour-diffusion method. A diffraction data set was collected to 1.50 Å resolution from a single crystal grown in 25% PEG 1500, 200 mM sodium thiocyanate pH 6.9, 10 mM phenol and 10% ethylene glycol. The crystal belonged to space group P212121, with unit-cell parameters a = 35.04, b = 50.32, c = 61.18 Å. The Q4DV70 structure was solved by molecular replacement using protein disulfide isomerase from yeast (PDB code 2b5e) as a search model. Initial refinement of the model indicated that the solution was correct. These data are being used for refinement of the model of Q4DV70.
doi:10.1107/S1744309109017734
PMCID: PMC2688432  PMID: 19478453
Q4DV70; Trypanosoma cruzi; thioredoxin domains

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