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Experimental methods for phase determination /MIR,MAD,SAD/ in crystallography of biological macromolecules
Brynda, Jiří
Importance of unbiased phases in post genomic era is emphasized. Article describes the classical methods of phase determination: 1) Multiple Isomorphous replacement, heavy-atom derivative (MIR) 2) Multiple Anomalous Dispersion (MAD) Phasing 3) Single Anomalous Dispersion (SAD) Phasing The better your initial, experimental map is, the more model you will be able to build confidently into this unbiased map before proceeding to phase combination. MAD phases result from perfectly isomorphous data, and do not deteriorate at low resolution as do MIR phases. MIR phases, on the other hand, provide superior phasing power at low resolution resulting in better connectivity. Not surprising, the best maps are either MAD or combined MIR-MAD maps.
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Processing of Diffraction Data Obtained by Oscillation Method on Crystals of Biological Macromolecules
Brynda, Jiří
The components of monochromatic diffractometer suitable for data collection on crystals of biological macromolecules are described, as well as basics of rotation method. The optimal strategy for collecting X-ray diffraction data from macromolecular crystals is discussed. The geometric factors influencing the completeness of data are arising from the symmetry of the reciprocal lattice and from the experimental setup; they affect quantitatively the completeness of the measured set of reflections.
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A Phenylnorstatine Inhibitor Binding to HIV-1 Protease: Geometry, Protonation and Subsite-Pocket Interactions Analyzed at Atomic Resolution
Brynda, Jiří ; Řezáčová, Pavlína ; Fábry, Milan ; Hořejší, Magdalena ; Štouračová, Renata ; Sedláček, Juraj ; Souček, M. ; Hradílek, M. ; Lepšík, M. ; Konvalinka, J.
The x-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH2 has been determined at 1.03 A, the highest resolution so far reported for any HIV PR complex. The inhibiot shows subnanomolar Ki values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance developoment. The structure displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. The high resolution permits to assess the donor/acceptor realtions of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. Structural mechanism for the unimpaired ihnibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses.
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Structural basis of HIV-1 and HIV-2 protease inhibition bya monoclonal antibody
Řezáčová, Pavlína ; Lescar, J. ; Brynda, Jiří ; Fábry, Milan ; Bentley, G. A. ; Sedláček, Juraj
The murine monoclonal antibody 1696, produced by immunisation with the HIV-1 protease,inhibits the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates, with inhibitionconstants in the low nanomolar range. This antibody cross-reacts with peptides that include theN-terminus of the enzyme (residues 1-7), a region which is highly conserved in sequence amongdifferent viral strains and which, furthermore, is crucial for homodimerization to the activeenzymatic form.<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />We report here two crystal structures of a recombinant single-chain Fv fragment of mAb 1696,expressed in E. coli, as a complex with a cross-reactive peptides from the HIV-1 PR and theHIV-2 PR at 2.7 ? resolution and 1.9 ? resolution respectively. On the basis of the interactionsseen in the complex three-dimensional structures, the cross-reactivity between mAb 1696 withthe HIV-1 and HIV-2 protease and their N-terminal peptides can be explained. In addition, acandidate mechanism of HIV PR inhibition by mAb 1696 is proposed which may help thedesign of alternative HIV protease inhibitors, aimed at dissociating the homodimeric viral enzyme.
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Crystallographic study of an anti=carbonic anhydrase IX monoclonal antibody M75
Štouračová, Renata ; Závada, Jan ; Závadová, Zuzana ; Pastoreková, S. ; Brynda, Jiří ; Fábry, Milan ; Král, Vlastimil ; Hořejší, Magdalena ; Sedláček, Juraj
Carbonic anhydrase IX (CA IX) is a cell surface protein, strongly associated with certain types of human carcinomas. Structural study of a CA IX-binding monoclonal antibody (mAb) M75, complexed with its epitope peptide may contribute toward elucidation of the role of CA IX. Monoclonal antibody M75 was obtained and proved to react excellently with native and denaturated CA IX. Using synthetic oligopeptides, the epitope of mAb M75 was localized in the proteoglycan domain of CA IX, in the region of a tandem repeat and identified as amino acids PGEEDLP. The Fab fragment was obtained by papain cleavage. We obtained crystals of free Fab M75 and Fab M75 complexed with two different epitope peptides. The data set for Fab M75 was collected and the structure solving is underway.
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Preliminary Crystallographic Study of ç-D-Crystalline Obtained from Patient`s Eye
Brynda, Jiří ; Řezáčová, Pavlína ; Štouračová, Renata ; Ondráček, Jan ; Kmoch, B. ; Asfaw, B. ; Elleder, M. ; Bezouška, K. ; Filipec, M. ; Sedláček, Juraj
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