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1.  Energetics of 5-bromo-4-chloro-3-indolyl-α-d-mannose binding to the Parkia platycephala seed lectin and its use for MAD phasing 
The first crystal structure of a Mimosoideae lectin, Parkia platycephala has been solved by MAD phasing using 5-bromo-4-chloro-3-indolyl-α-d-mannose as an anomalous X-ray scatterer. This strategy may be useful for structure elucidation of novel lectins or when molecular replacement methods fail.
Parkia platycephala belongs to the most primitive group of Leguminosae plants. Its seed lectin is made up of three homologous β-prism repeats and exhibits binding specificity for mannose/glucose. The properties of the association between the lectin from P. platycephala seeds and monosaccharide ligands were analysed by isothermal titration calorimetry and surface plasmon resonance. The results are consistent with the lectin bearing three thermodynamically identical binding sites for mannose/glucose per monomer with dissociation constants in the millimolar range. Binding of each ligand by the lectin is enthalpically driven. Crystals have been obtained of the lectin in complex with a brominated derivative of mannose (5-bromo-4-chloro-3-indolyl-α-d-mannose), which were suitable for deriving an electron-density map by MAD phasing. In agreement with the thermodynamic data, six Br atoms were found in the asymmetric unit of the monoclinic P21 crystals, which contained two P. platycephala lectin molecules. The availability of other Br derivatives of monosaccharides (glucose, galactose, fucose) may make this strategy widely useful for structure elucidation of novel lectins or when (as in the case of the P. platycephala lectin) molecular-replacement methods fail.
doi:10.1107/S1744309105004835
PMCID: PMC1952276  PMID: 16511032
protein–carbohydrate interactions; Parkia platycephala lectin; isothermal titration calorimetry; surface plasmon resonance; β-prism domain; MAD phasing
2.  Crystallization and preliminary X-ray analysis of a novel Kunitz-type kallikrein inhibitor from Bauhinia bauhinioides  
Crystallization and preliminary X-ray diffraction studies are reported for a novel Kunitz-type protease inhibitor from B. bauhinioides which contains no disulfide bridges.
A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293 K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87 Å resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P212121, with unit-cell parameters a = 46.70, b = 64.14, c = 59.24 Å. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (V M = 2.5 Å3 Da−1). Iodinated crystals were prepared and a derivative data set was also collected at 2.1 Å resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5 M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method.
doi:10.1107/S1744309105028496
PMCID: PMC1991323  PMID: 16511193
Kunitz-type kallikrein inhibitors; disulfide bridges; Bauhinia bauhinioides
3.  Purification, crystallization and preliminary X-ray analysis of Enterococcus casseliflavus aminoglycoside-2′′-phosphotransferase-IVa 
Aminoglycoside-2′′-phosphotransferase-IVa [APH(2′′)-IVa] is an enzyme that is responsible for high-level gentamicin resistance in E. casseliflavus isolates. Three different crystals of wild-type substrate-free APH(2′′)-IVa have been prepared and preliminary X-ray diffraction experiments have been undertaken on all three crystal forms.
The deactivation of aminoglycoside antibiotics by chemical modification is one of the major sources of bacterial resistance to this family of therapeutic compounds, which includes the clinically relevant drugs streptomycin, kanamycin and gentamicin. The aminoglycoside phosphotransferases (APHs) form one such family of enzymes responsible for this resistance. The gene encoding one of these enzymes, aminoglycoside-2′′-phosphotransferase-IVa [APH(2′′)-IVa] from Enterococcus casseliflavus, has been cloned and the protein (comprising 306 amino-acid residues) has been expressed in Escherichia coli and purified. The enzyme was crystallized in three substrate-free forms. Two of the crystal forms belonged to the orthorhombic space group P212121 with similar unit-cell parameters, although one of the crystal forms had a unit-cell volume that was approximately 13% smaller than the other and a very low solvent content of around 38%. The third crystal form belonged to the monoclinic space group P21 and preliminary X-ray diffraction analysis was consistent with the presence of two molecules in the asymmetric unit. The orthorhombic crystal forms of apo APH(2′′)-IVa both diffracted to 2.2 Å resolution and the monoclinic crystal form diffracted to 2.4 Å resolution; synchrotron diffraction data were collected from these crystals at SSRL (Stanford, California, USA). Structure determination by molecular replacement using the structure of the related enzyme APH(2′′)-IIa is proceeding.
doi:10.1107/S1744309109050039
PMCID: PMC2805544  PMID: 20057078
aminoglycoside-2′′-phosphotransferase-IVa; Enterococcus casseliflavus; antibiotic resistance
4.  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
5.  Superoxide reductase from Nanoarchaeum equitans: expression, purification, crystallization and preliminary X-ray crystallographic analysis 
Diffraction-quality crystals of N. equitans neelaredoxin have been produced. The expression, purification and crystallization of the protein and preliminary X-ray crystallographic analysis of the crystals are reported.
Superoxide reductases (SORs) are the most recent oxygen-detoxification system to be identified in anaerobic and microaerobic bacteria and archaea. SORs are metalloproteins that are characterized by their possession of a catalytic nonhaem iron centre in the ferrous form coordinated by four histidine ligands and one cysteine ligand. Ignicoccus hospitalis, a hyperthermophilic crenarchaeon, is the only organism known to date to serve as a host for Nanoarchaeum equitans, a nanosized hyperthermophilic archaeon isolated from a submarine hot vent which completely depends on the presence of and contact with I. hospitalis cells for growth to occur. Similarly to I. hospitalis, N. equitans has a neelaredoxin (a 1Fe-type SOR) that keeps toxic oxygen species under control, catalysing the one-electron reduction of superoxide to hydrogen peroxide. Blue crystals of recombinant N. equitans SOR in the oxidized form (12.7 kDa, 109 residues) were obtained using polyethylene glycol (PEG 2000 MME) as precipitant. These crystals diffracted to 1.9 Å resolution at 100 K and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 51.88, b = 82.01, c = 91.30 Å. Cell-content analysis suggested the presence of four monomers in the asymmetric unit. The Matthews coefficient (V M) was determined to be 1.9 Å3 Da−1, corresponding to an estimated solvent content of 36%. Self-rotation function and native Patterson calculations suggested a tetramer with 222 point-group symmetry, similar to other 1Fe-SORs. The three-dimensional structure will be determined by the molecular-replacement method.
doi:10.1107/S1744309111009432
PMCID: PMC3087648  PMID: 21543869
superoxide reductases; Nanoarchaeum equitans; oxidative stress; neelaredoxins
6.  Crystallization and preliminary X-ray crystallographic studies of succinic semialdehyde dehydrogenase from Streptococcus pyogenes  
Succinic semialdehyde dehydrogenase (SSADH) from S. pyogenes was purified and crystallized. Crystals of native and NAD+-complexed SSADH diffracted to resolutions of 1.6 and 1.7 Å, respectively.
Succinic semialdehyde dehydrogenase (SSADH) plays a critical role in the metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and catalyzes the NAD(P)+-coupled oxidation of succinic semialdehyde (SSA) to succinic acid (SA). SSADH from Streptococcus pyogenes has been purified and crystallized as the apoenzyme and in a complex with NAD+. The crystals of native and NAD+-complexed SSADH diffracted to resolutions of 1.6 and 1.7 Å, respectively, using a synchrotron-radiation source. Both crystals belonged to the orthorhombic space group P21212­1, with unit-cell parameters a = 93.3, b = 100.3, c = 105.1 Å for the native crystal and a = 93.3, b = 100.3, c = 105.0 Å for the complex crystal. Preliminary molecular replacement confirmed the presence of one dimer in both crystals, corresponding to a Matthews coefficient (V M) of 2.37 Å3 Da−1 and a solvent content of 48.0%.
doi:10.1107/S1744309111052055
PMCID: PMC3310532  PMID: 22442224
succinic semialdehyde dehydrogenase; NAD+; Streptococcus pyogenes; GabD
7.  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
8.  Expression, purification, crystallization and preliminary X-ray diffraction analysis of grass carp β2-microglobulin 
Grass carp β2-microglobulin was expressed in E. coli, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method with PEG 2K as precipitant. The crystals obtained belong to the orthorhombic space group P212121, with unit-cell parameters a = 38.72, b = 40.65, c = 71.12 Å.
β2-Microglobulin (β2m) is an essential subunit of MHC I molecules; it stabilizes the structure of MHC I and plays a pivotal role in coreceptor recognition. To date, structures of β2m have been solved for three different mammals: human, mouse and cattle. In order to illuminate the molecular evolutionary origin of β2m, an understanding of its structure in lower vertebrates becomes important. Here, grass carp (Ctenopharyngodon idellus) β2m (Ctid-β2m) was expressed, purified and crystallized. Diffraction data were collected to a resolution of 2.5 Å. The crystal belongs to space group P212121, with unit-cell parameters a = 38.72, b = 40.65, c = 71.12 Å. The Matthews coefficient and the solvent content were calculated to be 2.56 Å Da−1 and 52.07%, respectively, for one molecule per asymmetric unit. The structure has been solved by molecular replacement using monomeric human β2m as a model.
doi:10.1107/S1744309107068388
PMCID: PMC2374163  PMID: 18323608
β2-microglobulin; MHC I
9.  Preliminary X-ray crystallographic analysis of β-­carbonic anhydrase psCA3 from Pseudomonas aeruginosa  
Two crystal forms of β-carbonic anhydrase psCA3 from P. aeruginosa were grown. Crystal form A belonged to space group P212121, with unit-cell parameters a = 81.9, b = 84.9, c = 124.2 Å, and diffracted X-rays to 2.9 Å resolution; crystal form B belonged to space group P21212, with unit-cell parameters a = 69.9, b = 77.7, c = 88.5 Å, and diffracted X-rays to 3.0 Å resolution.
Pseudomonas aeruginosa is a Gram-negative bacterium that causes life-threatening infections in susceptible individuals and is resistant to most clinically available antimicrobials. Genomic and proteomic studies have identified three genes, pa0102, pa2053 and pa4676, in P. aeruginosa PAO1 encoding three functional β-carbonic anhydrases (β-CAs): psCA1, psCA2 and psCA3, respectively. These β-CAs could serve as novel antimicrobial drug targets for this pathogen. X-ray crystallographic structural studies have been initiated to characterize the structure and function of these proteins. This communication describes the production of two crystal forms (A and B) of β-CA psCA3. Form A diffracted to a resolution of 2.9 Å; it belonged to space group P212121, with unit-cell parameters a = 81.9, b = 84.9, c = 124.2 Å, and had a calculated Matthews coefficient of 2.23 Å3 Da−1 assuming four molecules in the crystallographic asymmetric unit. Form B diffracted to a resolution of 3.0 Å; it belonged to space group P21212, with unit-cell parameters a = 69.9, b = 77.7, c = 88.5 Å, and had a calculated Matthews coefficient of 2.48 Å3 Da−1 assuming two molecules in the crystallographic asymmetric unit. Preliminary molecular-replacement solutions have been determined with the PHENIX AutoMR wizard and refinement of both crystal forms is currently in progress.
doi:10.1107/S1744309113017594
PMCID: PMC3729167  PMID: 23908036
carbonic anhydrases; β-class carbonic anhydrases; Pseudomonas aeruginosa
10.  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
11.  Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion protein 
The crystallization of a specific nanobody against mouse PrPC and preliminary diffraction analysis of a crystal that diffracted to 1.23 Å resolution are presented.
Prion disorders are infectious diseases that are characterized by the conversion of the cellular prion protein PrPC into the pathogenic isoform PrPSc. Specific antibodies that interact with the cellular prion protein have been shown to inhibit this transition. Recombinant VHHs (variable domain of dromedary heavy-chain antibodies) or nanobodies are single-domain antibodies, making them the smallest antigen-binding fragments. A specific nanobody (Nb_PrP_01) was raised against mouse PrPC. A crystallization condition for this recombinant nanobody was identified using high-throughput screening. The crystals were optimized using streak-seeding and the hanging-drop method. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 30.04, b = 37.15, c = 83.00 Å, and diffracted to 1.23 Å resolution using synchrotron radiation. The crystal structure of this specific nanobody against PrPC together with the known PrPC structure may help in understanding the PrPC/PrPSc transition mechanism.
doi:10.1107/S1744309110042168
PMCID: PMC2998374  PMID: 21139215
Camelidae; nanobodies; prions; PrPC; PrPSc
12.  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
13.  Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands 
Crystals of the wild-type haloalkane dehalogenase DhaA derived from R. rhodochrous NCIMB 13064 and of its catalytically inactive variant DhaA13 were grown in the presence of various ligands and diffraction data were collected to high and atomic resolution.
Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon–halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P212121 as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively.
doi:10.1107/S1744309110051286
PMCID: PMC3034621  PMID: 21301099
haloalkane dehalogenases; DhaA; Rhodococcus rhodochrous; microseeding; atomic resolution
14.  Crystallization and preliminary X-ray crystallographic analysis of a helicase-like domain from a tomato mosaic virus replication protein 
Crystals of the helicase domain from a tomato mosaic virus replication protein obtained using the hanging-drop vapour-diffusion method at 285 K diffracted X-rays to 2.05 Å resolution. They belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.8, b = 128.3, c = 40.7 Å.
Tomato mosaic virus belongs to the genus Tobamovirus in the alphavirus-like superfamily of positive-strand RNA viruses. The alphavirus-like superfamily includes many plant and animal viruses of agronomical and clinical importance. These viruses encode replication-associated proteins that contain a putative superfamily 1 helicase domain. No three-dimensional structures for this domain have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of the 130K helicase domain are reported. Diffraction data were collected and processed to 2.05 and 1.75 Å resolution from native and selenomethionine-labelled crystals, respectively. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.8, b = 128.3, c = 40.7 Å.
doi:10.1107/S174430911104231X
PMCID: PMC3232162  PMID: 22139189
tomato mosaic virus; replicase protein; helicase domain
15.  Purification, crystallization and preliminary X-ray analysis of PPD6, a PsbP-domain protein from Arabidopsis thaliana  
The PsbP-domain-containing protein PPD6 from A. thaliana was expressed, purified and crystallized.
The PsbP protein is an extrinsic component of photosystem II that together with PsbO and PsbQ forms the thylakoid lumenal part of the oxygen-evolving complex in higher plants. In addition to PsbP, the thylakoid lumen contains two PsbP-like proteins (PPLs) and six PsbP-domain proteins (PPDs). While the functions of the PsbP-like proteins PPL1 and PPL2 are currently under investigation, the function of the PsbP-domain proteins still remains completely unknown. PPD6 is unique among the PsbP family of proteins in that it contains a conserved disulfide bond which can be reduced in vitro by thioredoxin. The crystal structure determination of the PPD6 protein has been initiated in order to elucidate its function and to gain deeper insights into redox-regulation pathways in the thylakoid lumen. PPD6 has been expressed, purified and crystallized and preliminary X-ray diffraction data have been collected. The crystals belonged to space group P21, with unit-cell parameters a = 47.0, b = 64.3, c = 62.0 Å, β = 94.2°, and diffracted to a maximum d-spacing of 2.1 Å.
doi:10.1107/S1744309111042023
PMCID: PMC3310529  PMID: 22442221
photosynthesis; thylakoid lumen; PsbP domain
16.  Crystallization and X-ray diffraction studies of arginine kinase from the white Pacific shrimp Litopenaeus vannamei  
The crystallization and preliminary X-ray diffraction analysis at 1.25 Å resolution of free-ligand arginine kinase from the Pacific whiteleg shrimp Litopenaeus vannamei are reported. Crystals belong to space group P212121, phases were determined by molecular replacement and refinement was performed with Phenix.
Crystals of an unligated monomeric arginine kinase from the Pacific whiteleg shrimp Litopenaeus vannamei (LvAK) were successfully obtained using the microbatch method. Crystallization conditions and preliminary X-ray diffraction analysis to 1.25 Å resolution are reported. Data were collected at 100 K on NSLS beamline X6A. The crystals belonged to space group P212121, with unit-cell parameters a = 56.5, b = 70.2, c = 81.7 Å. One monomer per asymmetric unit was found, with a Matthews coefficient (V M) of 2.05 Å3 Da−1 and 40% solvent content. Initial phases were determined by molecular replacement using a homology model of LvAK as the search model. Refinement was performed with PHENIX, with final R work and R free values of 0.15 and 0.19, respectively. Biological analysis of the structure is currently in progress.
doi:10.1107/S1744309112020180
PMCID: PMC3388921  PMID: 22750864
arginine kinases; Litopenaeus vannamei
17.  Purification, crystallization and preliminary X-ray characterization of a haemagglutinin from the seeds of Jatropha curcas  
A novel haemagglutinin from Jatropha curcas seeds is purified and crystallized. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P212121 and the crystals diffracted to 2.8 Å resolution at 103 K.
The plant Jatropha curcas (Euphorbiaceae) is an important source of biofuel from the inedible oil present in its toxic seeds. The toxicity arises from the presence of curcin, a ribosome-inactivating protein showing haemagglutination activity. In this communication, the purification, crystallization and preliminary X-ray characterization are reported of a small protein isolated from J. curcas seeds with a molecular mass of ∼10 kDa that agglutinates rabbit erythrocytes. The protein was crystallized using the hanging-drop vapour-diffusion method and also by the microbatch method in 72-well HLA plates, using PEG 8000 as the precipitant in both conditions. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P212121. The crystals diffracted to 2.8 Å resolution at 103 K.
doi:10.1107/S1744309111038218
PMCID: PMC3232132  PMID: 22139159
Jatropha curcas; haemagglutinins
18.  Crystallization and preliminary structure determination of the membrane-bound complex cytochrome c nitrite reductase from Desulfovibrio vulgaris Hildenborough 
The cytochrome c nitrite reductase complex from D. vulgaris Hildenborough has been crystallized. The preliminary crystallographic structure reveals a 2:1 NrfA:NrfH complex stoichiometry.
The cytochrome c nitrite reductase (cNiR) isolated from Desulfovibrio vulgaris Hildenborough is a membrane-bound complex formed of NrfA and NrfH subunits. The catalytic subunit NrfA is a soluble pentahaem cytochrome c that forms a physiological dimer of about 120 kDa. The electron-donor subunit NrfH is a membrane-anchored tetrahaem cytochrome c of about 18 kDa molecular weight and belongs to the NapC/NirT family of quinol dehydrogenases, for which no structures are known. Crystals of the native cNiR membrane complex, solubilized with dodecylmaltoside detergent (DDM), were obtained using PEG 4K as precipitant. Anomalous diffraction data were measured at the Swiss Light Source to 2.3 Å resolution. Crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 79.5, b = 256.7, c = 578.2 Å. Molecular-replacement and MAD methods were combined to solve the structure. The data presented reveal that D. vulgaris cNiR contains one NrfH subunit per NrfA dimer.
doi:10.1107/S1744309106016629
PMCID: PMC2243080  PMID: 16754983
cytochrome c nitrite reductase; membrane-bound NrfHA complex; NapC/NirT family; multi-haem protein
19.  Crystallization and preliminary X-ray analysis of molecular chaperone-like diol dehydratase-reactivating factor in ADP-bound and nucleotide-free forms 
The molecular chaperone-like reactivating factor for adenosylcobalamin (coenzyme B12) dependent diol dehydratase was crystallized in ADP-bound and nucleotide-free forms. Preliminary X-ray analysis indicated that crystals are orthorhombic and diffract to 2.0 Å.
Adenosylcobalamin (coenzyme B12) dependent diol dehydratase (EC 4.2.1.28) catalyzes the conversion of 1,2-diols and glycerol to the corresponding aldehydes. It undergoes mechanism-based inactivation by glycerol. The diol dehydratase-reactivating factor (DDR) reactivates the inactivated holoenzymes in the presence of adenosylcobalamin, ATP and Mg2+ by mediating the release of a damaged cofactor. This molecular chaperone-like factor was overexpressed in Escherichia coli, purified and crystallized in the ADP-bound and nucleotide-free forms by the sandwich-drop vapour-diffusion method. The crystals of the ADP-bound form belong to the orthorhombic system, with space group P212121 and unit-cell parameters a = 83.26, b = 84.60, c = 280.09 Å, and diffract to 2.0 Å. In the absence of nucleotide, DDR crystals were orthorhombic, with space group P212121 and unit-cell parameters a = 81.92, b = 85.37, c = 296.99 Å and diffract to 3.0 Å. Crystals of both forms were suitable for structural analysis.
doi:10.1107/S1744309105015733
PMCID: PMC1952328  PMID: 16511107
reactivating factor; molecular chaperone; diol dehydratase; adenosylcobalamin; coenzyme B12
20.  Expression, purification, crystallization and structure determination of two glutathione S-transferase-like proteins from Shewanella oneidensis  
The production and purification of recombinant SoGST3 and SoGST6, two GST-like proteins from S. oneidensis, are reported and preliminary crystallographic studies of crystals of the recombinant enzymes are presented.
Genome analysis of Shewanella oneidensis, a Gram-negative bacterium with an unusual repertoire of respiratory and redox capabilities, revealed the presence of six glutathione S-transferase-like genes (sogst1–sogst6). Glutathione S-­transferases (GSTs; EC 2.5.1.18) are found in all kingdoms of life and are involved in phase II detoxification processes by catalyzing the nucleophilic attack of reduced glutathione on diverse electrophilic substrates, thereby decreasing their reactivity. Structure–function studies of prokaryotic GST-like proteins are surprisingly underrepresented in the scientific literature when compared with eukaryotic GSTs. Here, the production and purification of recombinant SoGST3 (SO_1576) and SoGST6 (SO_4697), two of the six GST-like proteins in S. oneidensis, are reported and preliminary crystallographic studies of crystals of the recombinant enzymes are presented. SoGST3 was crystallized in two different crystal forms in the presence of GSH and DTT that diffracted to high resolution: a primitive trigonal form in space group P31 that exhibited merohedral twinning with a high twin fraction and a primitive monoclinic form in space group P21. SoGST6 yielded primitive orthorhombic crystals in space group P212121 from which diffraction data could be collected to medium resolution after application of cryo-annealing protocols. Crystal structures of both SoGST3 and SoGST6 have been determined based on marginal search models by maximum-likelihood molecular replacement as implemented in the program Phaser.
doi:10.1107/S1744309108014589
PMCID: PMC2496851  PMID: 18540073
glutathione S-transferases; Shewanella oneidensis
21.  Crystallization and preliminary X-ray analysis of the human respiratory syncytial virus nucleocapsid protein 
Crystals of the nucleocapsid protein of human respiratory syncytial virus have been obtained in two forms, the better of which diffracted to 3.6 Å resolution and contained ten subunits in the asymmetric unit.
Human respiratory syncytial virus (HRSV) has a nonsegmented negative-stranded RNA genome which is encapsidated by the HRSV nucleocapsid protein (HRSVN) that is essential for viral replication. HRSV is a common cause of respiratory infection in infants, yet no effective antiviral drugs to combat it are available. Recent data from an experimental anti-HRSV compound, RSV-604, indicate that HRSVN could be the target site for drug action. Here, the expression, purification and preliminary data collection of decameric HRSVN as well as monomeric N-terminally truncated HRSVN mutants are reported. Two different crystal forms of full-length seleno­methionine-labelled HRSVN were obtained that diffracted to 3.6 and ∼5 Å resolution and belonged to space group P212121, with unit-cell parameters a = 133.6, b = 149.9, c = 255.1 Å, and space group P21, with unit-cell parameters a = 175.1, b = 162.6, c = 242.8 Å, β = 90.1°, respectively. For unlabelled HRSVN, only crystals belonging to space group P21 were obtained that diffracted to 3.6 Å. A self-rotation function using data from the orthorhombic crystal form confirmed the presence of tenfold noncrystallographic symmetry, which is in agreement with a reported electron-microscopic reconstruction of HRSVN. Monomeric HRSVN generated by N-terminal truncation was designed to assist in structure determination by reducing the size of the asymmetric unit. Whilst such HRSVN mutants were monomeric in solution and crystallized in a different space group, the size of the asymmetric unit was not reduced.
doi:10.1107/S1744309108031059
PMCID: PMC2581706  PMID: 18997331
HRSVN; human respiratory syncytial virus; nucleocapsid proteins
22.  Purification, crystallization and preliminary X-ray diffraction data of UDP-galactopyranose mutase from Aspergillus fumigatus  
The cloning, overexpression, purification, crystallization and preliminary X-ray diffraction data are described for UDP-galactopyranose mutase, an enzyme involved in cell-wall synthesis in A. fumigatus.
Aspergillus fumigatus UDP-galactopyranose mutase (AfUGM) is a potential drug target involved in the synthesis of the cell wall of this fungal pathogen. AfUGM was recombinantly produced in Escherichia coli, purified and crystallized by the sitting-drop method, producing orthorhombic crystals that diffracted to a resolution of 3.25 Å. The crystals contained four molecules per asymmetric unit and belonged to space group P212121, with unit-cell parameters a = 127.72, b = 134.30, c = 173.84 Å. Incorporation of selenomethionine was achieved, but the resulting crystals did not allow solution of the phase problem.
doi:10.1107/S1744309112017915
PMCID: PMC3370916  PMID: 22684076
UDP-galactopyranose mutase; Aspergillus fumigatus
23.  Cloning, expression, purification, crystallization and preliminary X-ray analysis of Mycoplasma genitalium protein MG289 
Single orthorhombic crystals of M. genitalium protein MG289 have been grown and shown to diffract X-rays to 2.8 Å resolution with good statistics. The structure obtained from these data will help to provide insight into the function of the protein as well as improving the understanding of its role in this human pathogen.
Mycoplasma genitalium is a human pathogen that is associated with non­gonococcal urethritis in men and cervicitis in women. The cloning, expression, purification and crystallization of the protein MG289 from M. genitalium strain G37 are reported here. Crystals of MG289 diffracted X-rays to 2.8 Å resolution. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 49.7, b = 90.9, c = 176.1 Å. The diffraction data after processing had an overall R merge of 8.7%. The crystal structure of Cypl, the ortholog of MG289 from M. hyorhinis, has recently been determined, providing a reasonable phasing model; molecular replacement is currently under way.
doi:10.1107/S1744309109030565
PMCID: PMC2795598  PMID: 19724130
Mycoplasma genitalium; MG289; p37; extracytoplasmic thiamine-binding lipoprotein; Cypl
24.  Crystallization and preliminary crystallographic characterization of three peptidic inhibitors in complex with α-thrombin 
Human α-thrombin was crystallized in complex with specific peptide inhibitors of general sequence d-Phe-Pro-d-Arg-P1′-CONH2. The crystals belonged to the orthorhombic space group P212121 and diffracted to beyond 1.3 Å resolution.
The serine protease thrombin plays a major role in thrombosis and haemostasis. This has driven interest in thrombin inhibitors as potential antithrombotic drugs. Here, the crystallization and preliminary crystallographic analysis of human α-­thrombin in complex with three noncovalent peptide inhibitors of the general sequence d-Phe-Pro-d-Arg-P1′-CONH2 are reported. The crystals belonged to the orthorhombic space group P212121 and diffracted to beyond 1.3 Å resolution.
doi:10.1107/S1744309110043472
PMCID: PMC3079972  PMID: 21206024
thrombin; blood coagulation; peptide inhibitors; noncovalent
25.  Purification, crystallization and preliminary X-ray characterization of the human GTP fucose pyrophosphorylase 
The human GTP fucose pyrophosphohydrolase protein has been crystallized via the hanging-drop technique over a reservoir of polyethylene glycol (MW 8000) and ethylene glycol. The orthorhombic crystals diffract to 2.8 Å resolution.
The human nucleotide-sugar metabolizing enzyme GTP fucose pyrophosphorylase (GFPP) has been purified to homogeneity by an affinity chromatographic procedure that utilizes a novel nucleoside analog. This new purification regime results in a protein preparation that produces significantly better crystals than traditional purification methods. The purified 66.6 kDa monomeric protein has been crystallized via hanging-drop vapor diffusion at 293 K. Crystals of the native enzyme diffract to 2.8 Å and belong to the orthorhombic space group P212121. There is a single GFPP monomer in the asymmetric unit, giving a Matthews coefficient of 2.38 Å3 Da−1 and a solvent content of 48.2%. A complete native data set has been collected as a first step in determining the three-dimensional structure of this enzyme.
doi:10.1107/S1744309106008529
PMCID: PMC2222559  PMID: 16582493
GTP fucose pyrophosphohydrolase

Results 1-25 (360136)