X-ray diffraction analysis
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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 P2(1)2(1)2(1) 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.
- MeSH
- 2-propanol MeSH
- bakteriální proteiny chemie MeSH
- difrakce rentgenového záření MeSH
- hydrolasy chemie genetika metabolismus MeSH
- hydrolýza MeSH
- izoenzymy chemie genetika MeSH
- katalýza MeSH
- krystalizace MeSH
- krystalografie rentgenová metody MeSH
- ligandy MeSH
- propan analogy a deriváty MeSH
- Rhodococcus enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
X-ray structure analysis results have been widely used in the pharmaceutical research, development and control, especially for mapping polymorphism, to determine the chirality of active substances, in the pharmaceutical documentation and patent policy for many years. The greatest progress in X-ray diffraction techniques has been made in improving the quality of the poor material for successful data collection (magnetically oriented microcrystal arrays, serial snapshot crystallography). Prospects of the pharmaceutical application of X-ray crystallography lie in the acceleration of data collection, time-resolved structural studies obtained from the material of pharmaceutical batches without modification, and, in addition to that, in solving structures of semi-solid and amorphous phases and monitoring structural changes in drug formulations.
- MeSH
- difrakce rentgenového záření MeSH
- duševní vlastnictví MeSH
- farmaceutická technologie MeSH
- krystalografie rentgenová * metody přístrojové vybavení využití MeSH
- léčivé přípravky * analýza MeSH
- spektrální analýza metody přístrojové vybavení využití MeSH
- stereoizomerie MeSH
- Publikační typ
- práce podpořená grantem MeSH
The small bacterial laccase from the actinobacterium Streptomyces coelicolor which lacks the second of the three domains of the laccases structurally characterized to date was crystallized. This multi-copper phenol oxidase crystallizes in a primitive tetragonal lattice, with unit-cell parameters a = b = 179.8, c = 175.3 A. The crystals belong to either space group P4(1)2(1)2 or P4(3)2(1)2. The self-rotation function shows the presence of a noncrystallographic threefold axis in the structure. Phases will be determined from the anomalous signal of the natively present copper ions.
Galectin-4 is thought to play a role in the process of tumour conversion of cells of the alimentary tract and the breast tissue; however, its exact function remains unknown. With the aim of elucidating the structural basis of mouse galectin-4 (mGal-4) binding specificity, we have undertaken X-ray analysis of the N-terminal domain, CRD1, of mGal-4 in complex with lactose (the basic building block of known galectin-4 carbohydrate ligands). Crystals of CRD1 in complex with lactose were obtained using vapour-diffusion techniques. The crystals belong to tetragonal space group P42(1)2 with unit-cell parameters a = 91.1, b = 91.16, c = 57.10 A and preliminary X-ray diffraction data were collected to 3.2 A resolution. An optimized crystallization procedure and cryocooling protocol allowed us to extend resolution to 2.1 A. Structure refinement is currently under way; the initial electron-density maps clearly show non-protein electron density in the vicinity of the carbohydrate binding site, indicating the presence of one lactose molecule. The structure will help to improve understanding of the binding specificity and function of the potential colon cancer marker galectin-4.
- MeSH
- aminokyselinové motivy MeSH
- difrakce rentgenového záření MeSH
- financování organizované MeSH
- galektin 4 chemie metabolismus MeSH
- krystalizace MeSH
- laktosa chemie metabolismus MeSH
- ligandy MeSH
- myši MeSH
- nádorové biomarkery chemie metabolismus MeSH
- nádory tračníku chemie metabolismus MeSH
- peptidové fragmenty chemie metabolismus MeSH
- terciární struktura proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
Histidine-containing phosphotransfer proteins from Arabidopsis thaliana (AHP1-5) act as intermediates between sensor histidine kinases and response regulators in a signalling system called multi-step phosphorelay (MSP). AHP proteins mediate and potentially integrate various MSP-based signalling pathways (e.g. cytokinin or osmosensing). However, structural information about AHP proteins and their importance in MSP signalling is still lacking. To obtain a deeper insight into the structural basis of AHP-mediated signal transduction, the three-dimensional structure of AHP2 was determined. The AHP2 coding sequence was cloned into pRSET B expression vector, enabling production of AHP2 fused to an N-terminal His tag. AHP2 was expressed in soluble form in Escherichia coli strain BL21 (DE3) pLysS and then purified to homogeneity using metal chelate affinity chromatography and anion-exchange chromatography under reducing conditions. Successful crystallization in a buffer which was optimized for thermal stability yielded crystals that diffracted to 2.5 Å resolution.
- MeSH
- Arabidopsis metabolismus MeSH
- difrakce rentgenového záření MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fosfotransferasy chemie izolace a purifikace MeSH
- krystalizace MeSH
- proteiny huseníčku chemie izolace a purifikace MeSH
- signální transdukce * MeSH
- tranzitní teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The HsdR subunit of the type I restriction-modification system EcoR124I is responsible for the translocation as well as the restriction activity of the whole complex consisting of the HsdR, HsdM and HsdS subunits, and while crystal structures are available for the wild type and several mutants, the C-terminal domain comprising approximately 150 residues was not resolved in any of these structures. Here, three fusion constructs with the GFP variant pHluorin developed to overexpress, purify and crystallize the C-terminal domain of HsdR are reported. The shortest of the three encompassed HsdR residues 887-1038 and yielded crystals that belonged to the orthorhombic space group C2221, with unit-cell parameters a = 83.42, b = 176.58, c = 126.03 Å, α = β = γ = 90.00° and two molecules in the asymmetric unit (VM = 2.55 Å(3) Da(-1), solvent content 50.47%). X-ray diffraction data were collected to a resolution of 2.45 Å.
- MeSH
- difrakce rentgenového záření MeSH
- Escherichia coli chemie enzymologie genetika MeSH
- exprese genu MeSH
- klonování DNA MeSH
- krystalizace MeSH
- krystalografie rentgenová MeSH
- plazmidy chemie metabolismus MeSH
- podjednotky proteinů chemie genetika metabolismus MeSH
- proteiny z Escherichia coli chemie genetika metabolismus MeSH
- rekombinantní fúzní proteiny chemie genetika metabolismus MeSH
- restrikční endonukleasy typu I chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- zelené fluorescenční proteiny chemie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
This paper deals with an investigation of ceramic archaeological finds with the use of in-situ X-ray fluorescence analysis. Firstly, three configurations of X-ray fluorescence analyzers constructed and used at the Czech Technical University in Prague are described and compared for use in a non-destructive survey of siliceous materials. Detection limits, depth of analysis, the relation of the analyzed area, the homogeneity of the samples, and variations in the element concentrations are discussed. Secondly, many shards of postmediaeval pottery from Southern Moravia are analyzed with X-ray fluorescence analysis and some of them also with electron microprobe analysis. Selected results are described.
- MeSH
- barvicí látky analýza MeSH
- dějiny středověku MeSH
- difrakce rentgenového záření metody MeSH
- keramika analýza MeSH
- rukopisy jako téma dějiny MeSH
- sochařství dějiny MeSH
- spektrometrie rentgenová emisní metody MeSH
- Check Tag
- dějiny středověku MeSH
- Publikační typ
- časopisecké články MeSH
- historické články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
