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1.  Purification, crystallization and preliminary X-ray crystallographic analysis of chitinase from Bacillus cereus NCTU2 
The crystallization of B. cereus chitinase is reported.
Chitinases (EC 3.2.1.14) are found in a broad range of organisms, including bacteria, fungi and higher plants, and play different roles depending on their origin. A chitinase from Bacillus cereus NCTU2 (ChiNCTU2) capable of hydrolyzing chitin as a carbon and nitrogen nutrient has been identified as a member of the family 18 glycoside hydrolases. ChiNCTU2 of molecular weight 36 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of chitinase crystals at 1.10 Å resolution, the crystal belongs to space group P21, with unit-cell parameters a = 50.79, b = 48.79, c = 66.87 Å, β = 99.31°. Preliminary analysis indicates there is one chitinase molecule in the asymmetric unit, with a solvent content of 43.4%.
doi:10.1107/S1744309106031423
PMCID: PMC2242883  PMID: 16946479
chitinase; Bacillus cereus NCTU2
2.  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
3.  Isolation, purification, crystallization and preliminary crystallographic studies of chitinase from tamarind (Tamarindus indica) seeds 
A 34 kDa chitinase from tamarind (T. indica) seeds was purified, crystallized and characterized using X-ray diffraction.
A protein with chitinase activity has been isolated and purified from tamarind (Tamarindus indica) seeds. N-terminal amino-acid sequence analysis of this protein confirmed it to be an ∼34 kDa endochitinase which belongs to the acidic class III chitinase family. The protein was crystallized by the vapour-diffusion method using PEG 4000. The crystals belonged to the tetragonal space group P41, with two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.6 Å.
doi:10.1107/S1744309109006472
PMCID: PMC2664755  PMID: 19342775
chitinases; Tamarindus indicus
4.  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
5.  Crystallization and preliminary crystallographic analysis of a chitinase from Clonostachys rosea  
A chitinase from the nematophagous fungus C. rosea was overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 1.8 Å resolution.
CrChi1 is a chitinase from the nematophagous fungus Clonostachys rosea that plays a role in the infection of nematodes. In order to resolve the crystal structure of CrChi1 and to gain a better understanding of its biological functions, recombinant CrChi1 was crystallized at 291 K using PEG 3350 and ammonium dihydrogen phosphate as precipitant and a 1.8 Å resolution X-ray data set was collected from a single flash-cooled crystal (100 K). The crystals belonged to space group P21, with unit-cell parameters a = 44.1, b = 71.7, c = 59.1 Å, α = γ = 90, β = 91.3°. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient and solvent content were calculated to be 2.45 Å3 Da−1 and 40%, respectively. To our knowledge, this is the first structure determination study of a chitinase from a nematophagous fungus.
doi:10.1107/S1744309109008379
PMCID: PMC2664767  PMID: 19342787
chitinases; Clonostachys rosea
6.  Purification, crystallization and preliminary X-ray crystallographic analysis of PBP4 from Listeria monocytogenes  
The crystallization of PBP4 from L. monocytogenes is reported.
Penicillin-binding proteins (PBPs), which catalyze peptidoglycan synthesis, have been extensively studied as a well established target of antimicrobial agents, including β-lactam derivatives. However, remarkable resistance to β-lactams has developed among pathogenic bacteria since the clinical use of penicillin began. Recently, the glycosyltransferase (GT) domain of class A PBPs has been proposed as an attractive target for antibiotic development as moenomycin-bound GT-domain structures have been determined. In this study, a class A PBP4 from Listeria monocytogenes was overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystal belonged to the primitive orthorhombic space group P21212, with unit-cell parameters a = 84.6, b = 127.8, c = 54.9 Å. The structural information will contribute to the further development of moenomycin-derived antibiotics possessing broad-spectrum activity.
doi:10.1107/S1744309111027400
PMCID: PMC3212374  PMID: 22102039
penicillin-binding proteins; Listeria monocytogenes
7.  Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment 
A Fab fragment of a monoclonal anti-parathyroid hormone-related protein antibody was prepared and its complex with parathyroid hormone-related protein was crystallized. X-ray diffraction data were collected to 2.0 Å resolution.
Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 Å, and diffracted to 2.0 Å resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.
doi:10.1107/S1744309109006216
PMCID: PMC2664753  PMID: 19342773
bone cancer; GPCR recognition; monoclonal antibody Fab fragment; parathyroid hormone-related protein
8.  Crystallization and preliminary X-ray diffraction analysis of the metalloregulatory protein DtxR from Thermoplasma acidophilum  
Orthorhombic crystals of DtxR from T. acidophilum have been obtained. X-ray data were collected to 1.8 Å resolution using synchrotron radiation.
The diphtheria toxin repressor (DtxR) is a metal-ion-dependent transcriptional regulator which regulates genes encoding proteins involved in metal-ion uptake to maintain metal-ion homeostasis. DtxR from Thermoplasma acidophilum was cloned and overexpressed in Escherichia coli. Crystals of N-terminally His-tagged DtxR were obtained by hanging-drop vapour diffusion and diffracted to 1.8 Å resolution. DtxR was crystallized at 296 K using polyethylene glycol 4000 as a precipitant. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 61.14, b = 84.61, c = 46.91 Å, α = β = γ = 90°. The asymmetric unit contained approximately one monomer of DtxR, giving a crystal volume per mass (V M) of 2.22 Å3 Da−1 and a solvent content of 44.6%.
doi:10.1107/S1744309111051700
PMCID: PMC3274395  PMID: 22297991
DtxR; transcriptional regulators; metalloregulatory proteins; Thermoplasma acidophilum
9.  Crystallization and preliminary X-ray crystallographic studies of an exo-β-d-glucosaminidase from Trichoderma reesei  
An exo-β-d-glucosaminidase from T. reesei (Gls93) has been crystallized by the hanging-drop vapour-diffusion method. Diffraction data have been collected using synchrotron radiation.
Chitosan is degraded to glucosamine (GlcN) by chitosanase and exo-β-d-glucosaminidase (GlcNase). GlcNase from Trichoderma reesei (Gls93) is a 93 kDa extracellular protein composed of 892 amino acids. The enzyme liberates GlcN from the nonreducing end of the chitosan chain in an exo-type manner and belongs to glycoside hydrolase family 2. For crystallographic investigations, Gls93 was overexpressed in Pichia pastoris cells. The recombinant Gls93 had two molecular forms of ∼105 kDa (Gls93-F1) and ∼100 kDa (Gls93-F2), with the difference between them being caused by N-glycosylation. Both forms were crystallized by the hanging-drop vapour-diffusion method. Crystals of Gls93-F1 belonged to the orthorhombic space group P212121, with unit-cell parameters a = 98.27, b = 98.42, c = 108.28 Å, and diffracted to 1.8 Å resolution. Crystals of Gls93-F2 belonged to the ortho­rhombic space group P212121, with unit-cell parameters a = 67.84, b = 81.62, c = 183.14 Å, and diffracted to 2.4 Å resolution. Both crystal forms were suitable for X-ray structure analysis at high resolution.
doi:10.1107/S1744309110000606
PMCID: PMC2833044  PMID: 20208168
exo-β-d-glucosaminidases; exochitosanases; Gls93; Trichoderma reesei
10.  Crystallization, data collection and phasing of two digestive lysozymes from Musca domestica  
The digestive lysozymes 1 and 2 from M. domestica were crystallized by vapour diffusion. The crystallographic data were processed to a maximum resolution of 1.9 Å in both cases.
Lysozymes are mostly known for their defensive role against bacteria, but in several animals lysozymes have a digestive function. Here, the initial crystallographic characterization of two digestive lysozymes from Musca domestica are presented. The proteins were crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate or PEG/2-propanol as the precipitant. X-ray diffraction data were collected to a maximum resolution of 1.9 Å using synchrotron radiation. The lysozyme 1 and 2 crystals belong to the monoclinic space group P21 (unit-cell parameters a = 36.52, b = 79.44, c = 45.20 Å, β = 102.97°) and the orthorhombic space group P21212 (unit-cell parameters a = 73.90, b = 96.40, c = 33.27 Å), respectively. The crystal structures were solved by molecular replacement and structure refinement is in progress.
doi:10.1107/S1744309106024201
PMCID: PMC2242927  PMID: 16880547
digestive lysozymes; Musca domestica
11.  Expression, purification, crystallization and preliminary X-ray diffraction analysis of the apo form of InsP5 2-K from Arabidopsis thaliana  
A mutated version of InsP5 2-K allows the production of crystals of the apo form and structure determination using X-ray crystallography.
Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5 2-K) is a key enzyme that catalyzes the synthesis of phytic acid (IP6) from inositol 1,3,4,5,6-pentakisphos­phate (IP5) and ATP. The first structure of IP5 2-K, that from Arabidopsis thaliana, has been solved previously; it only crystallized in the presence of inositol, either the substrate IP5 or the product IP6, and failed to crystallize in its free state (without inositol). Based on structural analysis, a point mutation of IP5 2-K (W129A) has been produced in order to overcome this limitation and obtain information about protein conformational changes upon substrate binding. Here, the production and crystallization of W129A IP5 2-K in its free state and with bound nucleotide is described. These crystals differed from the native crystals and belonged to the orthorhombic space group P21212, with unit-cell parameters a = 66.00, b = 68.23, c = 105.80 Å and a = 63.06, b = 71.80, c = 100.23 Å, respectively. The crystals diffracted to resolutions of 2.22 Å (apo) and 2.05 Å (nucleotide bound) using synchrotron radiation and contained one molecule per asymmetric unit. The structures have been determined using the molecular-replacement method and refinement is being undertaken.
doi:10.1107/S1744309112017307
PMCID: PMC3370915  PMID: 22684075
inositol kinases; inositol phosphate; phytic acid; IP6; IP5 2-K
12.  Purification, crystallization and preliminary X-ray diffraction of the C-terminal bromodomain from human BRD2 
The C-terminal bromodomain of human BRD2 was cloned, expressed, purified and crystallized. A complete diffraction data set has been collected to 1.80 Å resolution.
BRD2 is a bromodomain-containing BET-family protein that associates with acetylated histones throughout the cell cycle. Although the tertiary structures of the bromodomains involved in histone acetyl transfer are already known, the structures of the BET-type bromodomains, which are required for tight association with acetylated chromatin, are poorly understood. Here, the expression, purification and crystallization of the C-terminal bromodomain of human BRD2 are reported. The protein was crystallized by the sitting-drop vapour-diffusion method in the orthorhombic space group P21212, with unit-cell parameters a = 71.78, b = 52.60, c = 32.06 Å and one molecule per asymmetric unit. The crystal diffracted beyond 1.80 Å resolution using synchrotron radiation.
doi:10.1107/S1744309107028473
PMCID: PMC2335128  PMID: 17620725
acetylation; cell cycle; chromatin; gene expression; histones; transcription
13.  Crystallization and preliminary X-ray analysis of a family 19 glycosyl hydrolase from Carica papaya latex 
A chitinase isolated from the latex of the tropical species Carica papaya has been crystallized. The addition of N-acetyl-d-glucosamine to the crystallization solution has improved the diffraction quality resolution of the crystal to 1.8 Å resolution.
A chitinase isolated from the latex of the tropical species Carica papaya has been purified to homogeneity and crystallized. This enzyme belongs to glycosyl hydrolase family 19 and exhibits exceptional resistance to proteolysis. The initially observed crystals, which diffracted to a resolution of 2.0 Å, were improved through modification of the crystallization protocol. Well ordered crystals were subsequently obtained using N-acetyl-d-glucosamine, the mono­mer resulting from the hydrolysis of chitin, as an additive to the crystallization solution. Here, the characterization of a chitinase crystal that belongs to the monoclinic space group P21, with unit-cell parameters a = 69.08, b = 44.79, c = 76.73 Å, β = 95.33° and two molecules per asymmetric unit, is reported. Diffraction data were collected to a resolution of 1.8 Å. Structure refinement is currently in progress.
doi:10.1107/S1744309108007823
PMCID: PMC2376395  PMID: 18453704
Carica papaya; chitinases; family 19 glycosyl hydrolases
14.  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
15.  Expression, purification, crystallization and preliminary crystallographic analysis of chitinase A from Vibrio carchariae  
This article describes the high-level expression, purification and crystallization as well as preliminary X-ray diffraction study of a family 18 chitinase, chitinase A from V. carchariae.
Chitinase A of Vibrio carchariae was expressed in Escherichia coli M15 host cells as a 575-amino-acid fragment with full enzymatic activity using the pQE60 expression vector. The yield of the highly purified recombinant protein was approximately 70 mg per litre of bacterial culture. The molecular mass of the expressed protein was determined by HPLC/ESI–MS to be 63 770, including the hexahistidine tag. Crystals of recombinant chitinase A were grown to a suitable size for X-ray structure analysis in a precipitant containing 10%(v/v) PEG 400, 0.1 M sodium acetate pH 4.6 and 0.125 M CaCl2. The crystals belonged to the tetragonal space group P422, with two molecules per asymmetric unit and unit-cell parameters a = b = 127.64, c = 171.42 Å. A complete diffraction data set was collected to 2.14 Å resolution using a Rigaku/MSC R-AXIS IV++ detector system mounted on an RU-H3R rotating-anode X-ray generator.
doi:10.1107/S1744309105027831
PMCID: PMC1991324  PMID: 16511189
Vibrio carchariae; chitinase A
16.  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
17.  Crystallization and preliminary X-ray analysis of AzoR (azoreductase) from Escherichia coli  
The crystallization and preliminary X-ray analysis of AzoR (azoreductase) have been performed.
AzoR (azoreductase), an FMN-dependent NADH-azo compound oxidoreductase from Escherichia coli, has been crystallized in the presence of FMN by the sitting-drop vapour-diffusion method using 2-propanol as a precipitant. AzoR catalyzes the reductive cleavage of azo groups. The crystals were found to diffract X-rays to beyond 1.8 Å resolution using a synchrotron-radiation source. The crystals belonged to the tetragonal space group P42212, with unit-cell parameters a = b = 92.2, c = 51.9 Å. The crystals are expected to contain one subunit of the homodimer in the asymmetric unit (V M = 2.6 Å3 Da−1) and to have a solvent content of 51.6%. Data sets were also collected from heavy-atom derivatives for use in phasing. As a result, crystals soaked in a solution containing K2PtCl4 for 23 d were found to be reasonably isomorphous to the native crystals and the presence of Pt atoms could be confirmed. The data sets from the native crystals and the K2PtCl4-derivatized crystals are being evaluated for use in structure determination by single isomorphous replacement with anomalous scattering.
doi:10.1107/S1744309105007918
PMCID: PMC1952434  PMID: 16511052
azoreductases
18.  Crystallization and preliminary X-ray characterization of d-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi  
d-3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) from P. flagi has been crystallized by the hanging-drop method.
A recombinant form of d-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) from Pseudomonas fragi has been crystallized by the hanging-drop method using PEG 3000 as a precipitating agent. The crystals belong to the orthorhombic group P21212, with unit-cell parameters a = 64.3, b = 99.0, c = 110.2 Å. The crystals are most likely to contain two tetrameric subunits in the asymmetric unit, with a V M value of 3.29 Å3 Da−1. Diffraction data were collected to a 2.0 Å resolution using synchrotron radiation at the BL6A station of the Photon Factory.
doi:10.1107/S1744309104024741
PMCID: PMC1952369  PMID: 16508084
d-3-hydroxybutyrate dehydrogenase
19.  Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases. 
Journal of Bacteriology  1992;174(2):408-414.
The gene (chiD) encoding the precursor of chitinase D was found to be located immediately upstream of the chiA gene, encoding chitinase A1, which is a key enzyme in the chitinase system of Bacillus circulans WL-12. Sequencing analysis revealed that the deduced polypeptide encoded by the chiD gene was 488 amino acids long and the distance between the coding regions of the chiA and chiD genes was 103 bp. Remarkable similarity was observed between the N-terminal one-third of chitinase D and the C-terminal one-third of chitinase A1. The N-terminal 47-amino-acid segment (named ND) of chitinase D showed a 61.7% amino acid match with the C-terminal segment (CA) of chitinase A1. The following 95-amino-acid segment (R-D) of chitinase D showed 62.8 and 60.6% amino acid matches, respectively, to the previously reported type III-like repeating units R-1 and R-2 in chitinase A1, which were shown to be homologous to the fibronectin type III sequence. A 73-amino-acid segment (residues 247 to 319) located in the putative activity domain of chitinase D was found to show considerable sequence similarity not only to other bacterial chitinases and class III higher-plant chitinases but also to Streptomyces plicatus endo-beta-N-acetylglucosaminidase H and the Kluyveromyces lactis killer toxin alpha subunit. The evolutionary and functional meanings of these similarities are discussed.
Images
PMCID: PMC205731  PMID: 1729234
20.  Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1 
Isolated modules of mouse coactivator-associated arginine methyltransferase 1 encompassing the protein arginine N-methyltransferase catalytic domain have been overexpressed, purified and crystallized. X-ray diffraction data have been collected and have enabled determination of the structures by multiple isomorphous replacement using anomalous scattering.
Coactivator-associated arginine methyltransferase 1 (CARM1) plays a crucial role in gene expression as a coactivator of several nuclear hormone receptors and also of non-nuclear receptor systems. Its recruitment by the transcriptional machinery induces protein methylation, leading to chromatin remodelling and gene activation. CARM128–507 and two structural states of CARM1140–480 were expressed, purified and crystallized. Crystals of CARM128–507 belong to space group P6222, with unit-cell parameters a = b = 136.0, c = 125.3 Å; they diffract to beyond 2.5 Å resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM128–507 was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1140–480 belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 Å; they diffract to beyond 2.7 Å resolution and contain two monomers in the asymmetric unit. Crystals of CARM1140–480 in complex with S-­adenosyl-l-homocysteine belong to space P21212, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 Å; they diffract to beyond 2.6 Å resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1140–480 were solved by molecular-replacement techniques from the structure of CARM128–507.
doi:10.1107/S1744309107011785
PMCID: PMC2330207  PMID: 17401209
CARM1; gene expression
21.  Expression, purification, crystallization and preliminary X-ray diffraction analysis of Arabidopsis thaliana cyclophilin 38 (AtCyp38) 
Crystallization of Arabidopsis thaliana cyclophilin 38. The crystal diffracts X-rays to 2.5 Å resolution.
AtCyp38 is one of the highly divergent multidomain cyclophilins from Arabidopsis thaliana. A recombinant form of AtCyp38 (residues 83–437) was expressed in Escherichia coli and purified to homogeneity. The protein was crystallized using the vapour-batch technique with PEG 6000 and t-butanol as precipitants. Crystals of recombinant AtCyp38 diffracted X-rays to better than 2.5 Å resolution at 95 K using a synchrotron-radiation source. The crystal belongs to the C-centred orthorhombic space group C2221, with unit-cell parameters a = 58.2, b = 95.9, c = 167.5 Å, and contains one molecule in the asymmetric unit. The selenomethionine derivative of the AtCyp38 protein was overexpressed, purified and crystallized in the same space group and data were collected to 3.5 Å at the NSLS synchrotron. The structure is being solved by the MAD method.
doi:10.1107/S1744309105037681
PMCID: PMC1978155  PMID: 16511242
cyclophilins; PPIases; AtCyp38
22.  A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037. 
Journal of Bacteriology  1996;178(17):5065-5070.
The specificity of chitinase C-1 of Streptomyces griseus HUT 6037 for the hydrolysis of the beta-1,4-glycosidic linkages in partially acetylated chitosan is different from that of other microbial chitinases. In order to study the primary structure of this unique chitinase, the chiC gene specifying chitinase C-1 was cloned and its nucleotide sequence was determined. The gene encodes a polypeptide of 294 amino acids with a calculated size of 31.4 kDa. Comparison of the amino acid sequence of the deduced polypeptide with that of other proteins revealed a C-terminal catalytic domain displaying considerable sequence similarity to the catalytic domain of plant class I, II, and IV chitinases which form glycosyl hydrolase family 19. The N-terminal domain of the deduced polypeptide exhibits sequence similarity to substrate-binding domains of several microbial chitinases and cellulases but not to the chitin-binding domains of plant chitinases. The previously purified chitinase C-1 from S. griseus is suggested to be generated by proteolytic removal of the N-terminal chitin-binding domain and corresponds to the catalytic domain of the chitinase encoded by the chiC gene. High-performance liquid chromatography analysis of the hydrolysis products from N-acetyl chitotetraose revealed that chitinase C-1 catalyzes hydrolysis of the glycosidic bond with inversion of the anomeric configuration, in agreement with the previously reported inverting mechanism of plant class I chitinases. This is the first report of a family 19 chitinase found in an organism other than higher plants.
PMCID: PMC178299  PMID: 8752320
23.  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
24.  Isolation, purification, crystallization and preliminary crystallographic studies of a chitinase from Crocus vernus  
Crystals of a chitinase from C. vernus were monoclinic, belonging to space group C2 with unit-cell parameters a = 172.3, b = 37.1, c = 126.4 Å, β = 127°, and diffracted to 2.1 Å resolution.
A chitinase has been isolated and purified from Crocus vernus corms. N-­terminal amino-acid sequence analysis of the approximately 30 kDa protein showed 33% identity to narbonin, a seed protein from Vicia narbonensis L. The C. vernus chitinase was crystallized by the hanging-drop vapour-diffusion method using PEG 8000 as the main precipitant. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 172.3, b = 37.1, c = 126.4 Å, β = 127° and two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.1 Å.
doi:10.1107/S1744309110053698
PMCID: PMC3053159  PMID: 21393839
chitinases; Crocus vernus
25.  Crystallization and preliminary X-ray diffraction analysis of a lectin from Canavalia maritima seeds 
A lectin from C. maritima was crystallized using the vapour-diffusion method and crystals diffracted to 2.1 Å resolution. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%, refinement is in progress.
A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 Å resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P21212, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 Å. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way.
doi:10.1107/S1744309104029197
PMCID: PMC1952371  PMID: 16508099
lectins; Canavalia maritima

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