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1.  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
2.  Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of a major group 7 allergen, Der f 7, from the dust mite Dermatophagoides farinae  
The major group 7 allergen, Der f 7, from the dust mite Dermatophagoides farinae has been crystallized and diffracted X-rays to a resolution of 2.24 Å.
Der f 7 is a major group 7 allergen from the dust mite Dermatophagoides farinae that shows 86% sequence identity to the homologous allergen Der p 7 from D. pteronyssinus. Der f 7 was successfully overexpressed in an Escherichia coli expression system and purified to homogeneity using Ni–NTA affinity and size-exclusion column chromatography. SeMet-labelled Der f 7 was crystallized by the hanging-drop vapour-diffusion method using a reservoir solution consisting of 0.1 M bis-tris pH 7.4 and 28% polyethylene glycol monomethyl ether 2000 at 293 K. X-ray diffraction data were collected to 2.24 Å resolution using synchrotron radiation. The crystals belonged to the orthorhombic system, space group P212121, with unit-cell parameters a = 50.19, b = 58.67, c = 123.81 Å. Based on the estimated Matthews coefficient (2.16 Å3 Da−1), two molecules of Der f 7 could be present in the asymmetric unit of the crystal lattice.
doi:10.1107/S174430911103836X
PMCID: PMC3232152  PMID: 22139179
group 7 allergens; Der f 7; Dermatophagoides farinae
3.  Crystallization and preliminary X-ray crystallographic study of the extracellular domain of the 4-1BB ligand, a member of the TNF family 
The extracellular domain of the 4-1BB ligand fused with glutathione-S-transferase was expressed in Escherichia coli (Origami) and purified by using affinity and ion-exchange column chromatographic methods. Crystals of the 4-1BB ligand were obtained at 290 K by the hanging-drop vapour-diffusion method.
The 4-1BB ligand, a member of the tumour necrosis factor (TNF) family, is an important co-stimulatory molecule that plays a key role in the clonal expansion and survival of CD8+ T cells. Signalling through binding of the 4-1BB ligand and 4-­1BB has been reported to enhance CD8+ T-cell expansion and protect activated CD8+ T cells from death. The 4-1BB ligand is an integral protein expressed on activated antigen-presenting cells. The extracellular domain of the 4-1BB ligand fused with glutathione-S-transferase was expressed in Escherichia coli (Origami) and purified by using affinity and ion-exchange column chromatographic methods. Crystals of the 4-1BB ligand were obtained at 290 K by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from these crystals to 2.8 Å resolution and the crystals belong to space group C2, with unit-cell parameters a = 114.6, b = 73.8, c = 118.50 Å, β = 115.5°.
doi:10.1107/S1744309105039242
PMCID: PMC2150926  PMID: 16511253
TNF; 4-1BB ligand; T cells; co-stimulation
4.  Structure of Mesorhizobium loti arylamine N-acetyltransferase 1 
The crystal structure of a M. loti arylamine N-acetyltransferase 1 has been determined at 2.0 Å resolution.
The arylamine N-acetyltransferase (NAT) enzymes have been found in a broad range of both eukaryotic and prokaryotic organisms. The NAT enzymes catalyse the transfer of an acetyl group from acetyl Co-enzyme A onto the terminal nitrogen of a range of arylamine, hydrazine and arylhydrazine compounds. Recently, several NAT structures have been reported from different prokaryotic sources including Salmonella typhimurium, Mycobacterium smegmatis and Pseudomonas aeruginosa. Bioinformatics analysis of the Mesorhizobium loti genome revealed two NAT paralogues, the first example of multiple NAT isoenzymes in a eubacterial organism. The M. loti NAT 1 enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme was crystallized in 0.5 M Ca(OAc)2, 16% PEG 3350, 0.1 M Tris–HCl pH 8.5 using the sitting-drop vapour-diffusion method. A data set diffracting to 2.0 Å was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic spacegroup P212121, with unit-cell parameters a = 53.2, b = 97.3, c = 114.3 Å. The structure was refined to a final free-R factor of 24.8%. The structure reveals that despite low sequence homology, M. loti NAT1 shares the common fold as reported in previous NAT structures and exhibits the same catalytic triad of residues (Cys-His-Asp) in the active site.
doi:10.1107/S1744309104030659
PMCID: PMC1952398  PMID: 16508079
arylamine N-acetyltransferase 1
5.  Purification, crystallization, small-angle X-ray scattering and preliminary X-ray diffraction analysis of the SH2 domain of the Csk-homologous kinase 
The Src-homology 2 (SH2) domain of Csk-family protein tyrosine kinases acts as a conformational switch to regulate their catalytic activity, which in turn promotes the inhibition of their proto-oncogenic targets, the Src-family kinases. Here, the expression, purification, small-angle X-ray scattering and preliminary diffraction analysis of the SH2 domain of the Csk-homologous kinase is reported.
The C-terminal Src kinase (Csk) and Csk-homologous kinase (CHK) are endogenous inhibitors of the proto-oncogenic Src family of protein tyrosine kinases (SFKs). Phosphotyrosyl peptide binding to their Src-homology 2 (SH2) domains activates Csk and CHK, enhancing their ability to suppress SFK signalling; however, the detailed mechanistic basis of this activation event is unclear. The CHK SH2 was expressed in Escherichia coli and the purified protein was characterized as monomeric by synchrotron small-angle X-ray scattering in-line with size-exclusion chromatography. The CHK SH2 crystallized in 0.2 M sodium bromide, 0.1 M bis-Tris propane pH 6.5 and 20% polyethylene glycol 3350 and the best crystals diffracted to ∼1.6 Å resolution. The crystals belonged to space group P2, with unit-cell parameters a = 25.8, b = 34.6, c = 63.2 Å, β = 99.4°.
doi:10.1107/S1744309110053728
PMCID: PMC3053158  PMID: 21393838
Csk-homologous kinase; Src-homology 2 domains; enzyme inhibition; Src-family protein tyrosine kinases; cancer; small-angle X-ray scattering
6.  Crystallization of Mycobacterium smegmatis methionyl-tRNA synthetase in the presence of methionine and adenosine 
The expression, purification and crystallization of methionyl-tRNA synthetase from Mycobacterium smegmatis. The crystals diffracted to 2.1 Å.
Methionyl-tRNA synthetase (MetRS) from Mycobacterium smegmatis was recombinantly expressed in Escherichia coli and purified using Ni2+-affinity and size-exclusion chromatography. Crystals formed readily in the presence of the ligands methionine and adenosine. These two ligands are components of an intermediate in the two-step catalytic mechanism of MetRS. The crystals were produced using the vapour-diffusion method and a full data set to 2.1 Å resolution was collected from a single crystal. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 155.9, b = 138.9, c = 123.3 Å, β = 124.8°. The presence of three molecules in the asymmetric unit corresponded to a solvent content of 60% and a Matthews coefficient of 3.1 Å3 Da−1. Structure determination is in progress.
doi:10.1107/S1744309109016704
PMCID: PMC2688425  PMID: 19478446
methionyl-tRNA synthetase; MetRS; Mycobacterium smegmatis; methionine; adenosine
7.  Expression, purification and crystallization of VP4 protease from Tellina virus 1 
Limited proteolysis of a monomeric fraction of Tellina virus 1 VP4 protease leads to crystals that diffract to beyond 2.1 Å resolution.
Tellina virus 1 is an aquabirnavirus that was isolated from the sand-dwelling marine bivalve mollusc Tellina tenuis. The self-encoded protease viral protein 4 (VP4) processes its own polyprotein to yield the individual proteins VP2 and VP3 that are required for viral assembly. VP4 protease utilizes a serine–lysine catalytic dyad in its mechanism. A full-length VP4 construct was overexpressed in Escherichia coli and purified to homogeneity using nickel-affinity chromatography. Ion-exchange and size-exclusion chromatographic steps were utilized to isolate a monomeric fraction of the protein. The purified monomeric VP4 was subjected to limited proteolysis to yield crystallizable protein. Crystal growth was performed using the hanging-drop vapour-diffusion method and was carried out at room temperature (∼296 K). Hexagonal crystals grew in the presence of PEG 8000, ammonium sulfate and urea. These crystals diffracted to beyond 2.1 Å resolution and belonged to space group P6422, with unit-cell parameters a = 59.1, b = 59.1, c = 208.1 Å, one molecule in the asymmetric unit and a solvent content of 42%.
doi:10.1107/S1744309110048803
PMCID: PMC3079999  PMID: 21206051
viral proteases; serine–lysine catalytic dyad mechanism; birnaviruses
8.  Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate 
The Trypanosoma cruzi dihydroorotate dehydrogenase, a key enzyme in pyrimidine de novo biosynthesis and redox homeostasis, was crystallized in complex with its first reaction product, orotate.
Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 Å resolution using synchrotron radiation (λ = 0.900 Å). X-ray diffraction data were collected at 100 K and processed to 1.9 Å resolution with 98.2% completeness and an overall R merge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 Å. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (V M) of 2.2 Å3 Da−1 and a solvent content of 44%.
doi:10.1107/S174430910502659X
PMCID: PMC1991314  PMID: 16511183
dihydroorotate dehydrogenase; pyrimidine biosynthesis; Trypanosoma cruzi; fumarate reductase; redox homeostasis; structure-based drug design
9.  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
10.  Enhancement of the Structural Stability of Full-Length Clostridial Collagenase by Calcium Ions 
Applied and Environmental Microbiology  2012;78(16):5839-5844.
The clostridial collagenases G and H are multidomain proteins. For collagen digestion, the domain arrangement is likely to play an important role in collagen binding and hydrolysis. In this study, the full-length collagenase H protein from Clostridium histolyticum was expressed in Escherichia coli and purified. The N-terminal amino acid of the purified protein was Ala31. The expressed protein showed enzymatic activity against azocoll as a substrate. To investigate the role of Ca2+ in providing structural stability to the full-length collagenase H, biophysical measurements were conducted using the recombinant protein. Size exclusion chromatography revealed that the Ca2+ chelation by EGTA induced interdomain conformational changes. Dynamic light scattering measurements showed an increase in the percent polydispersity as the Ca2+ was chelated, suggesting an increase in protein flexibility. In addition to these conformational changes, differential scanning fluorimetry measurements revealed that the thermostability was decreased by Ca2+ chelation, in comparison with the thermal melting point (Tm). The melting point changed from 54 to 49°C by the Ca2+ chelation, and it was restored to 54°C by the addition of excess Ca2+. These results indicated that the interdomain flexibility and the domain arrangement of full-length collagenase H are reversibly regulated by Ca2+.
doi:10.1128/AEM.00808-12
PMCID: PMC3406112  PMID: 22685155
11.  Purification and crystallization of yeast Ent1 ENTH domain 
The yeast Epsin-1 (ent1) gene has been cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals.
Members of the Epsin protein family regulate the ubiquitin/clathrin-dependent trafficking of transmembrane proteins. The yeast Epsin-1 (ent1) gene was cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals. X-ray analysis revealed that the crystallographic symmetry is primitive orthorhombic, space group P222, with unit-cell parameters a = 32.7, b = 35.5, c = 110.6 Å and a diffraction limit of 2.3 Å. Matthews coefficient calculations suggested that the crystal contained only the ENTH domain. This was corroborated by Coomassie Blue-stained SDS–PAGE analysis of dissolved crystals.
doi:10.1107/S1744309112022488
PMCID: PMC3388931  PMID: 22750874
Epsin; ENTH domain; ubiquitylation/clathrin-dependent endocytosis
12.  Expression, purification, crystallization and preliminary X-ray analysis of carbonyl reductase S1 from Candida magnoliae  
Carbonyl reductase S1 from C. magnoliae was expressed, purified and crystallized. The crystals obtained diffracted X-rays to 1.90 Å resolution and belonged to space group P6122 or P6522.
The NADPH-dependent carbonyl reductase S1 from Candida magnoliae stereoselectively catalyzes the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE), which is a chiral compound valuable as a building block for pharmaceuticals. Carbonyl reductase S1 was expressed in Escherichia coli and purified by Ni-affinity, ion-exchange and size-exclusion chromatography. Crystals of carbonyl reductase S1 were obtained by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. X-ray diffraction data were collected to 1.90 Å resolution using a synchrotron-radiation source. The crystals belonged to space group P6122 or P6522, with unit-cell parameters a = b = 77.7, c = 307.5 Å. The asymmetric unit contained two molecules of the protein, with a solvent content of 44.2%.
doi:10.1107/S1744309112011645
PMCID: PMC3374508  PMID: 22691783
stereoselectivity; short-chain dehydrogenase/reductases; carbonyl reductase S1; Candida magnoliae
13.  Towards structural studies of the old yellow enzyme homologue SYE4 from Shewanella oneidensis and its complexes at atomic resolution 
Of the four old yellow enzyme homologues found in S. oneidensis, SYE4 is the homologue most implicated in resistance to oxidative stress. SYE4 was recombinantly expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method.
Shewanella oneidensis is an environmentally versatile Gram-negative γ-proteo­bacterium that is endowed with an unusually large proteome of redox proteins. Of the four old yellow enzyme (OYE) homologues found in S. oneidensis, SYE4 is the homologue most implicated in resistance to oxidative stress. SYE4 was recombinantly expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the ortho­rhombic space group P212121 and were moderately pseudo-merohedrally twinned, emulating a P422 metric symmetry. The native crystals of SYE4 were of exceptional diffraction quality and provided complete data to 1.10 Å resolution using synchrotron radiation, while crystals of the reduced enzyme and of the enzyme in complex with a wide range of ligands typically led to high-quality complete data sets to 1.30–1.60 Å resolution, thus providing a rare opportunity to dissect the structure–function relationships of a good-sized enzyme (40 kDa) at true atomic resolution. Here, the attainment of a number of experimental milestones in the crystallographic studies of SYE4 and its complexes are reported, including isolation of the elusive hydride–Meisenheimer complex.
doi:10.1107/S1744309109050386
PMCID: PMC2805545  PMID: 20057079
SYE4; Shewanella oneidensis; old yellow enzyme homologues
14.  Expression, purification, crystallization and preliminary X-ray analysis of the KaiC-like protein PH0187 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 
The KaiC-like protein PH0187 from the hyperthermophilic archaeon P. horikoshii OT3 was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal of PH0187 diffracted X-rays to 2.75 Å resolution.
KaiC is the central protein in the circadian rhythm in cyanobacteria. The 28 kDa KaiC-like protein PH0187 from the hyperthermophilic archaeon Pyrococcus horikoshii was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of PH0187 were obtained using a reservoir solution consisting of 1.0 M ammonium phosphate monobasic and 0.1 M sodium citrate tribasic pH 5.3 (the final pH value of the reservoir solution was 4.8) and diffracted X-rays to 2.75 Å resolution. The crystal of PH0187 belonged to space group P6322, with unit-cell parameters a = b = 239.1, c = 106.5 Å. The crystal contained four PH0187 molecules in the asymmetric unit.
doi:10.1107/S1744309110048426
PMCID: PMC3079995  PMID: 21206047
PH0187; KaiC; circadian rhythm
15.  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
16.  Crystallization and preliminary X-ray diffraction analysis of vaccinia virus H1L phosphatase 
A catalytically inactive mutant of the dual-specificity phosphatase H1L from vaccinia virus was expressed recombinantly, purified and crystallized by the microbatch method. The crystals belong to the tetragonal space group P422 and diffraction data were collected to 2.1 Å resolution using a synchrotron-radiation source. Attempts to derivatize these crystals with xenon gas lead to a space-group change to I422 with a smaller unit cell and a diffraction limit of 3.0 Å.
The cysteine-based protein phosphatase H1L was the first reported dual-specificity protein phosphatase. H1L is encapsidated within the vaccinia virus and is required for successful host infection and for the production of viable vaccinia progeny. H1L has therefore been proposed as a target candidate for antiviral compounds. Recombinant H1L has been expressed in a catalytically inactive form using an Escherichia coli host, leading to purification and crystallization by the microbatch method. The crystals diffract to 2.1 Å resolution using synchrotron radiation. These crystals belong to space group P422, with unit-cell parameters a = b = 98.31, c = 169.15 Å, and are likely to contain four molecules in the asymmetric unit. A sulfur SAD data set was collected to 2.8 Å resolution on beamline BM14 at the ESRF to facilitate structure determination. Attempts to derivatize these crystals with xenon gas changed the space group to I422, with unit-cell parameters a = b = 63.28, c = 169.68 Å and a single molecule in the asymmetric unit. The relationship between these two crystal forms is discussed.
doi:10.1107/S1744309108003680
PMCID: PMC2374156  PMID: 18323605
vaccinia virus; dual-specificity phosphatase; cysteine-based phosphatase; poxvirus target
17.  Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of the catalytic domain of a hyperthermostable endo-1,4-β-d-mannanase from Thermotoga petrophila RKU-1 
The catalytic domain of a hyperthermostable endo-1,4-β-d-mannanase from T. petrophila RKU-1 has been cloned, overexpressed in E. coli cells, purified and crystallized in two distinct crystalline forms by the sitting-drop vapour-diffusion method.
Endo-1,4-β-d-mannanases play key roles in seed germination and fruit ripening and have recently received much attention owing to their potential applications in the food, detergent and kraft pulp industries. In order to delineate their structural determinants for specificity and stability, X-ray crystallographic investigations combined with detailed functional studies are being performed. In this work, crystals of the catalytic domain of a hyperthermostable endo-1,4-β-­d-mannanase from Thermotoga petrophila RKU-1 were obtained from three different conditions, resulting in two crystalline forms. Crystals from conditions with phosphate or citrate salts as precipitant (CryP) belonged to space group P212121, with unit-cell parameters a = 58.76, b = 87.99, c = 97.34 Å, while a crystal from a condition with ethanol as precipitant (CryE) belonged to space group I212121, with unit-cell parameters a = 91.03, b = 89.97, c = 97.89 Å. CryP and CryE diffracted to resolutions of 1.40 and 1.45 Å, respectively.
doi:10.1107/S1744309110029131
PMCID: PMC2935232  PMID: 20823531
endo-1,4-β-d-mannanases; thermostability; Thermotoga petrophila RKU-1
18.  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
19.  Cloning, purification, crystallization and preliminary X-ray analysis of ESX-1-secreted protein regulator (EspR) from Mycobacterium tuberculosis  
ESX-1 secreted protein regulator (EspR, Rv3849) from M. tuberculosis has been purified and crystallized, and diffracted to 3.2 Å resolution at wavelength 0.97625 Å.
ESX-1-secreted protein regulator (EspR; Rv3849) is a key regulator in Mycobacterium tuberculosis that delivers bacterial proteins into the host cell during infection. EspR binds directly to the Rv3616c-Rv3614c promoter and activates transcription and secretes itself from the bacterial cell by the ESX-1 system. The three-dimensional structure of EspR will aid in understanding the mechanisms by which it binds to the Rv3616c-Rv3614c promoter and is involved in transcriptional activation. This study will significantly aid in the development of EspR-based therapeutics against M. tuberculosis. The full-length EspR gene from M. tuberculosis (H37Rv strain) was cloned and overexpressed as a soluble protein in Escherichia coli. The protein was purified by affinity chromatography using His-tagged protein followed by size-exclusion chromatography. EspR was crystallized using polyethylene glycol 3350 as precipitant. The crystals diffracted to 3.2 Å resolution using synchrotron radiation of wavelength 0.97625 Å. The crystal belonged to space group P3121 and contained three monomers in the asymmetric unit. Native and heavy-atom-derivatized data sets were collected from EspR crystals for use in ab initio structure-solution techniques.
doi:10.1107/S174430911004618X
PMCID: PMC3079979  PMID: 21206031
ESX-1-secreted protein regulator; Rv3849; Mycobacterium tuberculosis
20.  Crystallization and preliminary X-ray analysis of crustacean hyperglycaemic hormone from the kuruma prawn Marsupenaeus japonicus in its weakly active precursor form 
Crustacean hyperglycaemic hormone from the kuruma prawn M. japonicus was crystallized by the sitting-drop vapour-diffusion method in its weakly active precursor form which has an extra glycine residue at the C-terminus. The crystals belonged to the orthorhombic space group P212121 and diffracted to 1.95 Å resolution.
Crustacean hyperglycaemic hormone (CHH) plays a pivotal role in the regulation of glucose metabolism in crustaceans. Pej-SGP-I, one of the six known CHHs in the kuruma prawn Marsupenaeus japonicus, was heterolo­gously expressed in Escherichia coli as an N-terminally His-tagged and Nus-tagged protein in its weakly active precursor form, Pej-SGP-I-Gly, which has an extra glycine residue at the C-terminus. The recombinant peptide was subjected to affinity purification, tag removal, further purification and crystallization by the sitting-drop vapour-diffusion method using NaCl as the main precipitant. The crystals diffracted to 1.95 Å resolution and the space group was assigned as primitive orthorhombic P212121, with unit-cell parameters a = 40.19, b = 53.65, c = 53.63 Å. The Matthews coefficient (V M = 1.73 Å3 Da−1) indicated that the crystal contained two Pej-SGP-I-Gly molecules per asymmetric unit, with a solvent content of 29.0%.
doi:10.1107/S1744309111040140
PMCID: PMC3232146  PMID: 22139173
Marsupenaeus japonicus; crustacean hyperglycaemic hormone
21.  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
22.  Crystallization and preliminary X-ray analysis of cryptochrome 3 from Arabidopsis thaliana  
Recombinant cryptochrome 3 from A. thaliana with FAD and MTHF cofactors has been crystallized using the hanging-drop vapour-diffusion technique in the orthorhombic space group P212121 and X-ray diffraction data were collected to 1.9 Å resolution.
Cryptochromes are flavoproteins which serve as blue-light receptors in plants, animals, fungi and prokaryotes and belong to the same protein family as the catalytically active DNA photolyases. Cryptochrome 3 from the plant Arabidopsis thaliana (cry3; 525 amino acids, 60.7 kDa) is a representative of the novel cryDASH subfamily of UV-A/blue-light receptors and has been expressed as a mature FAD-containing protein in Escherichia coli without the signal sequence that directs the protein into plant organelles. The purified cryptochrome was found to be complexed to methenyltetrahydrofolate as an antenna pigment. Crystals of the cryptochrome–antenna pigment complex were obtained by vapour diffusion and display orthorhombic symmetry, with unit-cell parameters a = 76.298, b = 116.782, c = 135.024 Å. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The asymmetric unit comprises a cry3 dimer, the physiological role of which remains to be elucidated.
doi:10.1107/S1744309105028897
PMCID: PMC1991327  PMID: 16511200
cryptochrome 3; light receptors
23.  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
24.  Expression, purification and preliminary crystallographic analysis of 2,4-dihydroxy-hepta-2-ene-1,7-dioate aldolase (HpcH) from Escherichia coli C 
2,4-Dihydroxy-hepta-2-ene-1,7-dioate aldolase from E. coli C has been purified and crystallized. Diffraction data were collected to 1.6 Å and structure determination by molecular replacement is in progress.
The gene encoding 2,4-dihydroxy-hepta-2-ene-1,7-dioate (HHED) aldolase (HpcH; EC 4.1.2) from Escherichia coli C was cloned into the high-expression plasmid pProEx-HTa and overexpressed in E. coli BL21 (DE3). The 28 kDa enzyme was purified using immobilized metal-affinity and size-exclusion chromatography prior to crystallization. Crystals were obtained by the hanging-drop vapour-diffusion method at 277 K from a number of screening conditions. Type I crystals grown in a solution containing 0.4 M ammonium dihydrogen phosphate belong to space group R32, with unit-cell parameters a = b = 128.92, c = 175.30 Å. Type II crystals grown in a solution containing 0.5 M sodium chloride, 0.1 M sodium citrate pH 5.5 belong to space group I222, with unit-cell parameters a = 133.39, b = 155.39, c = 168.80 Å. Complete data sets were collected to 1.6 and 2.0 Å from type I and type II crystals, respectively, using synchrotron radiation.
doi:10.1107/S1744309105023079
PMCID: PMC1978122  PMID: 16511168
aldolase; Escherichia coli; aromatic degradative pathway; 4-hydroxyphenylacetate
25.  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

Results 1-25 (429945)