X-ray crystal structure
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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
A mononuclear cadmium(II) complex of formula [Cd(5,5'-dmbipy)2(OAc)2]·2H2O (5,5'-dmbipy = 5,5'-dimethyl-2,2'-bipyridine and OAc = acetato ligand) has been synthesized and characterized by FT-IR, UV-Vis, 1H-NMR, elemental analysis and single-crystal X-ray structure analysis. The molecular structure of the complex shows a distorted tetragonal antiprism CdN4O4 coordination geometry around the cadmium atom, resulting in coordination by four nitrogen atoms from two 5,5'-dmbipy ligands and four oxygen atoms from two acetate anions. The interaction of this complex to FS-DNA (fish sperm DNA) has also been studied by electronic absorption, fluorescence and gel electrophoresis techniques. Binding constant (Kb), Stern-Volmer constant (Ksv), number of binding sites (n) and bimolecular quenching rate constant (kq) have been calculated from these spectroscopic data. These results have revealed that the metal complex can bind effectively to FS-DNA via groove binding. The calculated thermodynamic parameters (ΔH°, ΔS° and ΔG°) show that hydrogen bonding and van der Waals forces have an important function in the Cd(II) complex-DNA interaction. The antibacterial effects of the synthesized cadmium complex have also been examined in vitro against standard bacterial strains: one Gram-positive (Staphylococcus aureus, ATCC 25923) and one Gram-negative (Escherichia coli, ATCC 25922) bacteria, using disk diffusion and macro-dilution broth methods. The obtained results show that the Cd(II) complex exhibits a marked antibacterial activity which is significantly better than those observed for its free ligand and metal salt for both Gram-positive and Gram-negative bacteria. However, this metal complex is a more potent antibacterial agent against the Gram-positive than that of the Gram-negative bacteria.Communicated by Ramaswamy H. Sarma.
- MeSH
- algoritmy MeSH
- antibakteriální látky chemie farmakologie MeSH
- Bacteria účinky léků MeSH
- DNA chemie MeSH
- kadmium chemie MeSH
- krystalografie rentgenová MeSH
- ligandy MeSH
- magnetická rezonanční spektroskopie MeSH
- mikrobiální testy citlivosti MeSH
- molekulární modely * MeSH
- molekulární struktura MeSH
- pyridiny chemická syntéza chemie farmakologie MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- techniky syntetické chemie MeSH
- termodynamika MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The crystal structure of the N-terminal domain of the RNA polymerase δ subunit (Nδ) from Bacillus subtilis solved at a resolution of 2.0Å is compared with the NMR structure determined previously. The molecule crystallizes in the space group C222(1) with a dimer in the asymmetric unit. Importantly, the X-ray structure exhibits significant differences from the lowest energy NMR structure. In addition to the overall structure differences, structurally important β sheets found in the NMR structure are not present in the crystal structure. We systematically investigated the cause of the discrepancies between the NMR and X-ray structures of Nδ, addressing the pH dependence, presence of metal ions, and crystal packing forces. We convincingly showed that the crystal packing forces, together with the presence of Ni(2+) ions, are the main reason for such a difference. In summary, the study illustrates that the two structural approaches may give unequal results, which need to be interpreted with care to obtain reliable structural information in terms of biological relevance.
- MeSH
- Bacillus subtilis enzymologie MeSH
- DNA řízené RNA-polymerasy chemie MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů * MeSH
- krystalografie rentgenová metody MeSH
- nukleární magnetická rezonance biomolekulární metody MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally "dry," has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from Spinacia oleracea. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein.
- MeSH
- aminokyselinové motivy MeSH
- fotosystém II (proteinový komplex) chemie MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- Ramanova spektroskopie MeSH
- rostlinné proteiny chemie MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- Spinacia oleracea chemie MeSH
- terciární struktura proteinů MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The structure of the Fc fragment of monoclonal antibody IgG2b from hybridom M75 of Mus musculus has been determined by single crystal X-ray diffraction. This is the first report of the structure of the murine immunoglobulin isotype IgG2b. The structure refined at 2.1 A resolution provides more detailed structural information about native oligosaccharides than was previously available. High-quality Fourier maps provide a clear identification of alpha-l-fucose with partial occupancy in the first branch of the antennary oligosaccharides. A unique Fc:Fc interaction was observed at the C(H)2-C(H)3 interface.
- MeSH
- financování organizované MeSH
- glykosylace MeSH
- imunoglobulin G chemie MeSH
- imunoglobuliny - Fc fragmenty chemie MeSH
- imunokomplex chemie MeSH
- krystalizace MeSH
- krystalografie rentgenová metody MeSH
- monoklonální protilátky chemie MeSH
- myši MeSH
- oligosacharidy chemie MeSH
- sekundární struktura proteinů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
β-Mannosidase (EC 3.2.1.25) is an important exoglycosidase specific for the hydrolysis of terminal β-linked mannoside in various oligomeric saccharide structures. β-Mannosidase from Aspergillus niger was expressed in Pichia pastoris and purified to clear homogeneity. β-Mannosidase was crystallized in the presence of D-mannose and the crystal diffracted to 2.41 Å resolution. The crystal belonged to space group P1, with unit-cell parameters a=62.37, b=69.73, c=69.90 Å, α=108.20, β=101.51, γ=103.20°. The parameters derived from the data collection indicate the presence of one molecule in the asymmetric unit.
- MeSH
- Aspergillus niger chemie enzymologie MeSH
- beta-mannosidasa chemie genetika MeSH
- fungální proteiny chemie genetika MeSH
- krystalizace MeSH
- krystalografie rentgenová MeSH
- mannosa chemie MeSH
- Pichia chemie genetika MeSH
- rekombinantní proteiny chemie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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
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 A resolution. The crystals of native FerB belonged to space group P2(1), with unit-cell parameters a = 61.6, b = 110.1, c = 65.2 A, beta = 118.2 degrees and four protein molecules in the asymmetric unit, whilst the SeMet-substituted form crystallized in space group P2(1)2(1)2, with unit-cell parameters a = 61.2, b = 89.2, c = 71.5 A and two protein molecules in the asymmetric unit. Structure determination by the three-wavelength MAD/MRSAD method is now in progress.
Bacteriophage ϕ6 is a double-stranded RNA virus that has been extensively studied as a model organism. Here we describe structure determination of ϕ6 major capsid protein P1. The protein crystallized in base centered orthorhombic space group C2221. Matthews's coefficient indicated that the crystals contain from four to seven P1 subunits in the crystallographic asymmetric unit. The self-rotation function had shown presence of fivefold axes of non-crystallographic symmetry in the crystals. Thus, electron density map corresponding to a P1 pentamer was excised from a previously determined cryoEM reconstruction of the ϕ6 procapsid at 7 Å resolution and used as a model for molecular replacement. The phases for reflections at higher than 7 Å resolution were obtained by phase extension employing the fivefold non-crystallographic symmetry present in the crystal. The averaged 3.6 Å-resolution electron density map was of sufficient quality to allow model building.
- MeSH
- bakteriofág phi 6 chemie MeSH
- elektronová kryomikroskopie MeSH
- konformace proteinů MeSH
- krystalizace MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- virové plášťové proteiny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
