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1.  Crystallization and initial X-ray diffraction studies of the flavoenzyme NAD(P)H:(acceptor) oxidoreductase (FerB) from the soil bacterium Paracoccus denitrificans  
The flavin-dependent enzyme FerB from P. denitrificans has been purified and both native and SeMet-substituted FerB have been crystallized. The two variants crystallized in two different crystallographic forms belonging to the monoclinic space group P21 and the orthorhombic space group P21212, respectively. X-ray diffraction data were collected to 1.75 Å resolution for both forms.
The flavin-dependent enzyme FerB from Paracoccus denitrificans reduces a broad range of compounds, including ferric complexes, chromate and most notably quinones, at the expense of the reduced nicotinamide adenine dinucleotide cofactors NADH or NADPH. Recombinant unmodified and SeMet-substituted FerB were crystallized under similar conditions by the hanging-drop vapour-diffusion method with microseeding using PEG 4000 as the precipitant. FerB crystallized in several different crystal forms, some of which diffracted to approximately 1.8 Å resolution. The crystals of native FerB belonged to space group P21, with unit-cell parameters a = 61.6, b = 110.1, c = 65.2 Å, β = 118.2° and four protein molecules in the asymmetric unit, whilst the SeMet-substituted form crystallized in space group P21212, with unit-cell parameters a = 61.2, b = 89.2, c = 71.5 Å and two protein molecules in the asymmetric unit. Structure determination by the three-wavelength MAD/MRSAD method is now in progress.
doi:10.1107/S1744309110005099
PMCID: PMC2852337  PMID: 20383015
flavoenzymes; quinone reductases; Paracoccus denitrificans
2.  The purification and crystallization of mDia1 in complex with RhoC 
An N-terminal construct of mDia1, a member of the diaphanous-related formin family, was overexpressed, purified and crystallized in complex with RhoC. Data collection from native and SeMet crystals is reported.
An N-terminal construct of mouse mDia1 was recombinantly expressed in Escherichia coli, purified and crystallized in complex with truncated human RhoC using the hanging-drop vapour-diffusion method. Crystals were obtained using PEG 2K MME and MgSO4 as a precipitating agent and belong to the orthorhombic space group P21212, with unit-cell parameters a = 148.4, b = 85.2, c = 123.2 Å. Complete native and SeMet-derivative data sets were collected at 100 K to 3.0 and 3.4 Å resolution, respectively, using synchrotron radiation.
doi:10.1107/S1744309105001065
PMCID: PMC1952246  PMID: 16511001
mDia1; RhoC; GTPases; diaphanous-related formins
3.  Cloning, crystallization and preliminary X-ray study of XC1258, a CN-hydrolase superfamily protein from Xanthomonas campestris  
A CN-hydrolase superfamily protein from the plant pathogen X. campestris has been overexpressed in E. coli, purified and crystallized.
CN-hydrolase superfamily proteins are involved in a wide variety of non-peptide carbon–nitrogen hydrolysis reactions, producing some important natural products such as auxin, biotin, precursors of antibiotics etc. These reactions all involve attack on a cyano or carbonyl carbon by a conserved novel catalytic triad Glu-Lys-Cys through a thiol acylenzyme intermediate. However, classification into the CN-hydrolase superfamily based on sequence similarity alone is not straightforward and further structural data are necessary to improve this categorization. Here, the cloning, expression, crystallization and preliminary X-­ray analysis of XC1258, a CN-hydrolase superfamily protein from the plant pathogen Xanthomonas campestris (Xcc), are reported. The SeMet-substituted XC1258 crystals diffracted to a resolution of 1.73 Å. They are orthorhombic and belong to space group P21212, with unit-cell parameters a = 143.8, b = 154.63, c = 51.3 Å, respectively.
doi:10.1107/S1744309106035433
PMCID: PMC2225184  PMID: 17012795
CN hydrolase; nitrilase superfamily; Xanthomonas campestris; structural genomics
4.  Crystallization and preliminary crystallographic study of a recombinant predicted acetamidase/formamidase from the thermophile Thermoanaerobacter tengcongensis  
A predicated acetamidase/formanidase from the archaeon T. tengcongensis and its SeMet substitute have been crystallized and undergone preliminarily crystallographic studies including MAD data collection.
No crystal structures are yet available for homologues of a predicted acetamidase/formamidase (Amds/Fmds) from the archaeon Thermoanaerobacter tengcongensis. The Amds/Fmds gene was cloned and expressed as a soluble protein in Escherichia coli. Native Amds/Fmds and its SeMet-substituted form were purified and crystallized by vapour diffusion in hanging drops at 296 K. The native crystals, which were grown in PEG 8000, belong to the monoclinic space group P21, with unit-cell parameters a = 41.23 (3), b = 152.88 (6), c = 100.26 (7) Å, β = 99.49 (3)°. The diffraction data were collected to 2.00 Å resolution using synchrotron radiation. Based on a predicted solvent content of 50%, a Matthews coefficient of 2.44 Å3 Da−1 and two main peaks in the self-rotation function, the asymmetric unit is predicted to contain two dimers of the 32 kDa native protein. MAD data were collected for the SeMet protein, but the corresponding crystals display different unit-cell parameters and appear to contain four dimers in the asymmetric unit.
doi:10.1107/S1744309104030519
PMCID: PMC1952406  PMID: 16508105
acetamidase/formamidase; Thermoanaerobacter tengcongensis
5.  Expression, purification and preliminary crystallographic analysis of human thyroid hormone responsive protein 
Human Thrsp has been crystallized as a prelude to the determination of its three-dimensional structure by X-ray crystallography.
Thyroid hormone responsive protein (Thrsp, also known as Spot 14 and S14) is a carbohydrate-inducible and thyroid-hormone-inducible nuclear protein specific to liver, adipose and lactating mammary tissues. Thrsp functions to activate genes encoding fatty-acid synthesis enzymes. Recent studies have shown that in some cancers human Thrsp (hS14) localizes to the nucleus and is amplified, suggesting that it plays a role in the regulation of lipogenic enzymes during tumourigenesis. Thrsp, a member of the Spot 14 superfamily, is an acidic homodimeric protein with no sequence similarity to other mammalian gene products and its biochemical function is elusive. To shed light on the structure–function relationship of this protein, human Thrsp was crystallized. Recombinant human Thrsp (hThrsp), the N-terminally truncated human Thrsp10–146 (hThrsp9) and their selenomethionyl (SeMet) derivatives were expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Diffraction-quality crystals were grown at 293 K using Li2SO4 as a precipitant. Using synchrotron radiation, data for the hThrsp SeMet derivative, hThrsp9 and its SeMet derivative were collected to 4.0, 3.0 and 3.6 Å resolution, respectively, at 100 K. The crystals of full-length hThrsp and its SeMet derivative belonged to space group P41212, with approximate unit-cell parameters a = b = 123.9, c = 242.1 Å, α = β = γ = 90.0°. In contrast, the crystals of the truncated hThrsp9 and its SeMet derivative belonged to space group P212121, with approximate unit-cell parameters a = 91.6, b = 100.8, c = 193.7 Å, α = β = γ = 90.0°. A molecular-replacement solution calculated using a murine Spot 14 structure as a search model indicated the presence of six molecules per asymmetric unit, comprising three hThrsp homodimers.
doi:10.1107/S1744309111021099
PMCID: PMC3151134  PMID: 21821901
breast cancer; lipogenesis; Spot 14; S14; Thrsp
6.  Expression, purification, crystallization and preliminary crystallographic study of the SRA domain of the human UHRF1 protein 
The SET- and RING finger-associated (SRA) domain of human UHRF1 protein has been overexpressed in E. coli, purified and crystallized. The three-dimensional structure of the protein in its SeMet form was solved from 2.2 Å diffraction data using the multiple anomalous dispersion method.
Human UHRF1 belongs to the unique mammalian family of proteins which contain a SET- and RING finger-associated (SRA) domain. This 180-residue domain has been reported to play key roles in the functions of the protein. It allows UHRF1 to bind methylated DNA, histone deacetylase 1 and DNA methyltransferase 1, suggesting a bridge between DNA methylation and the histone code. No structural data is available for any SRA domain. Native and SeMet-labelled SRA domains of human UHRF1 were overexpressed in Escherichia coli cells, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. A complete MAD data set was collected to 2.2 Å resolution at 100 K. Crystals of the SeMet-labelled protein belonged to the trigonal space group P3221, with unit-cell parameters a = b = 53.78, c = 162.05 Å.
doi:10.1107/S1744309108027462
PMCID: PMC2564892  PMID: 18931436
SRA domains; UHRF1
7.  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
8.  Expression, purification, crystallization and preliminary X-ray analysis of maleylacetate reductase from Burkholderia sp. strain SJ98 
Purification and preliminary X-ray crystallographic analysis of maleylacetate reductase encoded by the pnpD gene is reported.
Maleylacetate reductase (EC 1.3.1.32) is an important enzyme that is involved in the degradation pathway of aromatic compounds and catalyzes the reduction of maleylacetate to 3-oxoadipate. The gene pnpD encoding maleylacetate reductase in Burkholderia sp. strain SJ98 was cloned, expressed in Escherichia coli and purified by affinity chromatography. The enzyme was crystallized in both native and SeMet-derivative forms by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant at 293 K. The crystals belonged to space group P21212, with unit-cell parameters a = 72.91, b = 85.94, c = 53.07 Å. X-ray diffraction data for the native and SeMet-derivative crystal were collected to 2.7 and 2.9 Å resolution, respectively.
doi:10.1107/S1744309109047319
PMCID: PMC2802890  PMID: 20054138
maleylacetate reductase; Burkholderia sp. strain SJ98
9.  Expression, purification, crystallization and preliminary crystallographic analysis of BipD, a component of the Burkholderia pseudomallei type III secretion system 
A construct consisting of residues 10–310 of mature BipD, a component of the B. pseudomallei type III secretion system, has been crystallized. Native BipD crystals and SeMet and K2PtCl4 derivative crystals have undergone preliminary crystallographic analysis.
A construct consisting of residues 10–310 of BipD, a component of the Burkholderia pseudomallei type III secretion system (T3SS), has been overexpressed as a GST fusion, cleaved from the GST tag and purified. Crystals were grown of native and selenomethionine-labelled BipD. The crystals grow in two different polymorphs from the same condition. The first polymorph belongs to space group C222, with unit-cell parameters a = 103.98, b = 122.79, c = 49.17 Å, a calculated Matthews coefficient of 2.4 Å3 Da−1 (47% solvent content) and one molecule per asymmetric unit. The second polymorph belongs to space group P21212, with unit-cell parameters a = 136.47, b = 89.84, c = 50.15 Å, and a calculated Matthews coefficient of 2.3 Å3 Da−1 (45% solvent content) for two molecules per asymmetric unit (analysis of the self-rotation function indicates the presence of a weak twofold non-crystallographic symmetry axis in this P21212 form). The native crystals of both forms give diffraction data to 2.7 Å resolution, while the SeMet-labelled P21212 crystals diffract to 3.3 Å resolution. A K2PtCl4 derivative of the P21212 form was also obtained and data were collected to 2.7 Å with radiation of wavelength λ = 0.933 Å. The Pt-derivative anomalous difference Patterson map revealed two self-peaks on the Harker sections.
doi:10.1107/S1744309106027035
PMCID: PMC1894745  PMID: 16946464
BipD; type III secretion system; Burkholderia pseudomallei
10.  Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus 
The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described.
White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-­terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P212121, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution.
doi:10.1107/S1744309107029351
PMCID: PMC2335124  PMID: 17620728
white spot syndrome virus; envelope proteins
11.  Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenes  
Native and selenomethionine-labelled FAD synthetase from C. ammoniagenes have been crystallized by the hanging-drop vapour-diffusion method. A MAD data set for SeMet-labelled FAD synthetase was collected to 2.42 Å resolution, while data sets were collected to 1.95 Å resolution for the native crystals.
FAD synthetase from Corynebacterium ammoniagenes (CaFADS), a prokary­otic bifunctional enzyme that catalyses the phosphorylation of riboflavin as well as the adenylylation of FMN, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. Diffraction-quality cubic crystals of native and selenomethionine-labelled (SeMet-CaFADS) protein belonged to the cubic space group P213, with unit-cell parameters a = b = c = 133.47 Å and a = b = c = 133.40 Å, respectively. Data sets for native and SeMet-containing crystals were collected to 1.95 and 2.42 Å resolution, respectively.
doi:10.1107/S1744309109044789
PMCID: PMC2802882  PMID: 20054130
FAD synthetase; Corynebacterium ammoniagenes
12.  Crystallization and preliminary X-ray diffraction analysis of GCIP/HHM transcriptional regulator 
Native and selenomethionine-derivatized crystals of full-length human GCIP/HHM protein were obtained. The crystals belonged to space group P3221 and the best native crystal diffracted to 3.5 Å resolution.
GCIP/HHM is a human nuclear protein that is implicated in regulation of cell proliferation. Its primary structure contains helix–loop–helix and leucine-zipper motifs but lacks a DNA-binding basic region. Native and selenomethionine-derivatized (SeMet) crystals of full-length GCIP/HHM were obtained using the hanging-drop vapour-diffusion method. The crystals were greatly improved by adding tris(2-carboxyethyl)phosphine as a reducing reagent and diffracted to 3.5 Å resolution. Preliminary phase calculations using the data set obtained from the SeMet crystal suggested that the crystal belonged to space group P3221 and contained one molecule per asymmetric unit. Structure determination by the multiple-wavelength anomalous dispersion method using the SeMet crystals is in progress.
doi:10.1107/S1744309108038219
PMCID: PMC2628855  PMID: 19153449
TGF-β; transcription factors; Id-family proteins; carcinogenesis
13.  Purification, crystallization and preliminary X-ray diffraction analysis of the CBS-domain pair from the Methanococcus jannaschii protein MJ0100 
The C-terminal domain of the Methanococcus jannaschii protein MJ0100 includes a CBS-domain pair and has been overexpressed, purified and crystallized. Crystals of selenomethionine-substituted (SeMet) protein were also grown.
CBS domains are small protein motifs consisting of a three-stranded β-sheet and two α-helices that are present in proteins of all kingdoms of life and in proteins with completely different functions. Several genetic diseases in humans have been associated with mutations in their sequence, which has made them promising targets for rational drug design. The C-terminal domain of the Methanococcus jannaschii protein MJ0100 includes a CBS-domain pair and has been overexpressed, purified and crystallized. Crystals of selenomethionine-substituted (SeMet) protein were also grown. The space group of both the native and SeMet crystals was determined to be orthorhombic P212121, with unit-cell parameters a = 80.9, b = 119.5, c = 173.3 Å. Preliminary analysis of the X-ray data indicated that there were eight molecules per asymmetric unit in both cases.
doi:10.1107/S1744309108027930
PMCID: PMC2564890  PMID: 18931440
CBS domains; Methanococcus jannaschii
14.  Crystallization and preliminary structure analysis of CobE, an essential protein of cobalamin (vitamin B12) biosynthesis 
P. aeruginosa CobE, a protein implicated in vitamin B12 biosynthesis, has been crystallized and data on the native and SeMet forms recorded to resolutions of 1.9 and 1.7 Å, respectively. The anomalous measurements will be used for phasing.
CobE, a protein implicated in vitamin B12 biosynthesis, from Pseudomonas aeruginosa has been overexpressed in Escherichia coli, purified and crystallized using hanging-drop vapour diffusion. The crystals belong to the primitive orthorhombic space group P212121, with unit-cell parameters a = 31.86, b = 41.07, c = 87.41 Å. The diffraction extends to a resolution of 1.9 Å. There is one molecule per asymmetric unit and the estimated solvent content is 35%. SeMet-labelled CobE has been prepared and crystallizes under the same conditions as the native protein with diffraction to 1.7 Å. The anomalous measurements will be used for phasing.
doi:10.1107/S1744309105006731
PMCID: PMC1952438  PMID: 16511064
CobE; vitamin B12 biosynthesis
15.  Crystallization and preliminary X-ray diffraction analysis of Bacillus subtilis YwfE, an l-amino-acid ligase 
Native and selenomethionine-derivatized (SeMet) crystals of Bacillus subtilis YwfE in the presence of ADP, MgCl2 and the dipeptide l-Ala-l-Gln were obtained using the hanging-drop vapour-diffusion method.
Bacillus subtilis YwfE, an l-amino-acid ligase, catalyzes the formation of an α-­dipeptide from l-amino acids in an ATP-dependent manner. In order to elucidate the substrate-recognition mode and the reaction mechanism of this ligase, native and selenomethionine-derivatized (SeMet) crystals of YwfE in the presence of ADP, MgCl2 and the dipeptide l-Ala-l-Gln were obtained using the hanging-drop vapour-diffusion method. These crystals diffracted to 1.9 and 2.8 Å resolution, respectively. Preliminary SAD phase calculations using the data set from the SeMet crystal suggested that the crystal belonged to the hexagonal space group P6522, with unit-cell parameters a = b = 90.85, c = 250.31 Å, and contained one molecule in the asymmetric unit with a solvent content of 57.3%.
doi:10.1107/S174430911105425X
PMCID: PMC3274404  PMID: 22298000
l-amino-acid ligases; ATP-grasp fold
16.  Crystallization and preliminary X-ray analysis of BigR, a transcription repressor from Xylella fastidiosa involved in biofilm formation 
In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method.
BigR (biofilm growth-associated repressor) is a novel repressor protein that regulates the transcription of an operon implicated in biofilm growth in both Xylella fastidiosa and Agrobacterium tumefaciens. This protein binds to a palindromic TA-rich element located in the promoter of the BigR operon and strongly represses transcription of the operon. BigR contains a helix–turn–helix (HTH) domain that is found in some members of the ArsR/SmtB family of metal sensors, which control metal resistance in bacteria. Although functional studies have suggested that BigR does not act as a metal sensor, the presence of two cysteines and a methionine in its primary structure raised the possibility of BigR being a metal-ligand protein. In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from native and SeMet crystals to resolutions of 1.95 and 2.2 Å, respectively. Both crystals belong to space group P321 and contain one molecule per asymmetric unit.
doi:10.1107/S1744309107028722
PMCID: PMC2335149  PMID: 17620720
BigR; transcription repressors; biofilm formation; Xylella fastidiosa
17.  Overexpression, purification, characterization and preliminary crystallographic study of ­phosphoglycolate phosphatase from Shigella flexneri 2a strain 301 
Recombinant phosphoglycolate phosphatase from S. flexneri was overexpressed, purified, characterized and crystallized using the hanging-drop vapour-diffusion method. SeMet-labelled protein was also prepared and was crystallized for phase determination using the MAD technique.
Phosphoglycolate phosphatase has a salvage function in the metabolism of the 2-­phosphoglycolate formed during bacterial DNA repair. In order to better understand its dimerization behaviour, the influence of metal ions on its activity and its catalytic mechanism at the molecular level, recombinant phosphoglyco­late phosphatase from Shigella flexneri was overexpressed, purified, characterized and crystallized by the hanging-drop vapour-diffusion method at 291 K using polyethylene glycol 3500 as a precipitant and zinc acetate as an additive. The crystals belonged to space group R3, with unit-cell parameters a = 88.1, b = 88.1, c = 259.2 Å, corresponding to the presence of two molecules in the asymmetric unit. SeMet-labelled protein was also prepared and crystallized for use in phase determination. Initial structure determination using the multiwave­length anomalous dispersion (MAD) method clearly revealed that SfPGPase bears an α-helical cap domain that differs from that of a previously reported orthologue.
doi:10.1107/S1744309108039067
PMCID: PMC2628850  PMID: 19153451
phosphoglycolate phosphatase; Shigella flexneri
18.  Mutation of High-Affinity Methionine Permease Contributes to Selenomethionyl Protein Production in Saccharomyces cerevisiae▿ †  
Applied and Environmental Microbiology  2010;76(19):6351-6359.
The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of Saccharomyces cerevisiae and the identification of a mutant allele of the MUP1 gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the mup1 mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the mup1 mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.
doi:10.1128/AEM.01026-10
PMCID: PMC2950440  PMID: 20693451
19.  Expression, purification, crystallization and preliminary diffraction data characterization of Escherichia coli ribonuclease II (RNase II) 
Diffraction data from E. coli RNase II crystals of wild type and of an inactive mutant and its SeMet-derivative form were obtained to 2.44 and 2.74 Å resolution, providing a set of preliminary phases. An improved purification protocol allowed higher reproducibility in the crystallization of the mutant form.
RNA degradation is important in the post-transcriptional control of gene expression. The processing, degradation and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a 643-amino-acid enzyme that degrades single-stranded RNA from its 3′-­end, releasing ribonucleoside 5′-monophosphates. RNase II was expressed both as the wild type and as a D209N mutant form. The latter was also produced as an SeMet derivative. The various protein forms were crystallized using the vapour-diffusion method. Wild-type RNase II was crystallized in two crystal forms, both of which belonged to space group P21. X-ray diffraction data were collected to 2.44 and 2.75 Å resolution, with unit-cell parameters a = 56.8, b = 125.7, c = 66.2 Å, β = 111.9° and a = 119.6, b = 57.2, c = 121.2 Å, β = 99.7°, respectively. The RNase II D209N mutant gave crystals that belonged to space group P65, with unit-cell parameters a = b = 86.3, c = 279.2 Å, and diffracted to 2.74 Å. Diffraction data from the mutant and its SeMet derivative enabled the determination of a partial Se-atom substructure by SIRAS.
doi:10.1107/S1744309106021506
PMCID: PMC2242952  PMID: 16820694
RNase II; RNA degradation
20.  Purification, crystallization and preliminary X-ray analysis of the PCNA2–PCNA3 complex from Sulfolobus tokodaii strain 7 
A PCNA2−PCNA3 complex which has recently been identified from S. tokodaii strain 7 was overexpressed, purified and crystallized in two crystal forms.
Crenarchaeal PCNA is known to consist of three subunits (PCNA1, PCNA2 and PCNA3) that form a heterotrimer (PCNA123). Recently, another heterotrimeric PCNA composed of only PCNA2 and PCNA3 was identified in Sulfolobus tokodaii strain 7 (stoPCNAs). In this study, the purified stoPCNA2–stoPCNA3 complex was crystallized by hanging-drop vapour diffusion. The crystals obtained belonged to the orthorhombic space groups I222 and P21212, with unit-cell parameters a = 91.1, b = 111.8, c = 170.9 Å and a = 91.1, b = 160.6, c = 116.6 Å, respectively. X-ray diffraction data sets were collected to 2.90 Å resolution for the I222 crystals and to 2.80 Å resolution for the P21212 crystals.
doi:10.1107/S1744309109044479
PMCID: PMC2802881  PMID: 20054129
PCNA; Sulfolobus tokodaii
21.  Purification, crystallization and preliminary X-ray diffraction analysis of human enolase-phosphatase E1 
Recombinant human E1 enzyme has been crystallized using the hanging-drop vapour-diffusion method and diffraction-quality crystals were grown at 291 K using PEG 4000 as precipitant.
Enolase-phosphatase E1 (MASA) is a bifunctional enzyme in the ubiquitous methionine-salvage pathway and catalyzes the continuous reaction of 2,3-­diketo-5-methylthio-1-phosphopentane to yield the acireductone metabolite. Recombinant human E1 enzyme has been crystallized using the hanging-drop vapour-diffusion method and diffraction-quality crystals were grown at 291 K using PEG 4000 as precipitant. Diffraction data were collected to 1.7 Å resolution from SeMet-derivative crystals at 100 K using synchrotron radiation. The crystals belong to space group P212121, with unit-cell parameters a = 54.02, b = 57.55, c = 87.32 Å. The structure was subsequently solved by the multi-wavelength anomalous diffraction (MAD) phasing method.
doi:10.1107/S174430910501184X
PMCID: PMC1952300  PMID: 16511085
enolase-phosphatase E1; methionine salvage
22.  Preliminary crystallographic analysis of sugar cane phosphoribosylpyrophosphate synthase 
X-ray diffraction data have been collected from crystals of recombinant sugar cane phosphoribosylpyrophosphate synthase (PRS) and analysis has revealed its quaternary structure, localizing this PRS into the class of enzymes forming an hexameric oligomer of 223 kDa.
Phosphoribosylpyrophosphate synthases (PRS; EC 2.7.6.1) are enzymes that are of central importance in several metabolic pathways in all cells. The sugar cane PRS enzyme contains 328 amino acids with a molecular weight of 36.6 kDa and represents the first plant PRS to be crystallized, as well as the first phosphate-independent PRS to be studied in molecular detail. Sugar cane PRS was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Using X-ray diffraction experiments it was determined that the crystals belong to the orthorhombic system, with space group P21212 and unit-cell parameters a = 213.2, b = 152.6, c = 149.3 Å. The crystals diffract to a maximum resolution of 3.3 Å and a complete data set to 3.5 Å resolution was collected and analysed.
doi:10.1107/S1744309104025825
PMCID: PMC1952407  PMID: 16508088
phosphoribosylpyrophosphate; PRPP synthase; sugar cane
23.  Crystallization and preliminary X-ray studies of azoreductases from Bacillus sp. B29 
In order to determine the structure–function relationship of the azo-dye reduction mechanism, an X-ray crystallographic study of azoreductases was performed. Selenomethionine-labelled AzrA (SeMet-AzrA) and AzrC were crystallized by the hanging-drop vapour-diffusion method.
Azoreductases from Bacillus sp. B29 are NADH-dependent flavoenzymes which contain a flavin mononucleotide (FMN) as a prosthetic group and exist as homodimers composed of 23 kDa subunits. These enzymes catalyze the reductive degradation of various azo compounds by a ping-pong mechanism. In order to determine the structure–function relationship of the azo-dye reduction mechanism, an X-ray crystallographic study of azoreductases was performed. Selenomethionine-labelled AzrA (SeMet-AzrA) and AzrC were crystallized by the hanging-drop vapour-diffusion method. A crystal of SeMet-AzrA diffracted to 2.0 Å resolution and was determined to belong to space group P212121, with unit-cell parameters a = 56.9, b = 69.0, c = 105.4 Å. The native crystals of AzrC belonged to space group C2, with unit-cell parameters a = 192.0, b = 56.6, c = 105.5 Å, β = 115.7°, and diffracted to 2.21 Å resolution.
doi:10.1107/S1744309110007785
PMCID: PMC2864677  PMID: 20445244
azoreductases; NADH-dependent oxidoreductases; flavoproteins; Bacillus sp. B29
24.  Crystallization and preliminary X-ray crystallographic analysis of the α-2,6-sialyltransferase PM0188 from Pasteurella multosida  
The α-2,6-sialyltransferase PM0188 from P. multocida was purified using affinity-column chromatographic methods and crystallized using the hanging-drop vapour-diffusion method at 293 K.
Sialyltransferase is an enzyme that transfers the sialic acid moiety from cytidine-5-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the carbohydrate group of various glycoproteins. These glycoproteins are involved in inflammation, embryogenesis, immune defence and metastasis of cancer cells by cell–cell interactions or cell–matrix interactions. The α-2,6-sialyltransferase PM0188 from Pasteurella multocida was purified using affinity-column chromatographic methods and crystallized using the hanging-drop vapour-diffusion method at 293 K. MAD data were collected to 1.9 Å resolution from an SeMet-substituted crystal. The crystal belongs to space group P21, with unit-cell parameters a = 52.9, b = 61.0, c = 64.6 Å, α = γ = 90, β = 112.3°. Assuming the presence of one molecule in the asymmetric unit, the solvent content is estimated to be about 45%.
doi:10.1107/S1744309106000844
PMCID: PMC2150949  PMID: 16511286
PM0188; sialyltransferases
25.  Expression, purification, crystallization and preliminary X-ray analysis of sepiapterin reductase from Chlorobium tepidum  
Sepiapterin reductase from C. tepidum has been crystallized by the hanging-drop vapour-diffusion method. Phasing information has been obtained experimentally by MAD using a SeMet derivative.
Sepiapterin reductase from Chlorobium tepidum (CT-SR) produces l-threo-tetrahydrobiopterin, an isomer of tetrahydrobiopterin, in the last step of de novo synthesis initiating from GTP. Native CT-SR and a selenomethionine (SeMet) derivative of CT-SR have been crystallized by the hanging-drop vapour-diffusion method using PEG 400 as precipitant. CT-SR crystals belong to space group R32, with unit-cell parameters a = b = 201.142, c = 210.184 Å, and contain four molecules in the asymmetric unit. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The structure of CT-SR has been determined using MAD phasing. There is one CT-SR tetramer in the asymmetric unit formed by two closely interacting CT-SR dimers. The solvent content is calculated to be about 67.2%.
doi:10.1107/S174430910403444X
PMCID: PMC1952253  PMID: 16510994
sepiapterin reductase; tetrahydrobiopterin; Chlorobium tepidum

Results 1-25 (1191417)