S1004 Methods for structural characterization of biomolecules

Přírodovědecká fakulta
podzim 2014
Rozsah
2/0. 2 kr. (plus ukončení). Doporučované ukončení: zk. Jiná možná ukončení: k.
Vyučující
prof. RNDr. Michaela Wimmerová, Ph.D. (přednášející)
Garance
prof. RNDr. Michaela Wimmerová, Ph.D.
Národní centrum pro výzkum biomolekul – Přírodovědecká fakulta
Dodavatelské pracoviště: Národní centrum pro výzkum biomolekul – Přírodovědecká fakulta
Omezení zápisu do předmětu
Předmět je otevřen studentům libovolného oboru.
Cíle předmětu
At the end of the course students should be able to: understand and explain principles of crystal structure determination of proteins;
solve protein structure
interpret electron density maps
analyze and validate pdb coordinates
Osnova
  • 1. Protein crystallography – crystallization Thermodynamic view of crystallization (entropy, enthalpy), crystallization dynamics (nucleation, crystal growth), phase diagram of crystallization, crystallization techniques (vapor diffusion, counter-diffusion, seeding techniques, under oil crystallization), high throughput methods. 2. Protein crystallography – physical background Molecular symmetry, X-ray scattering, diffraction theory (Bragg’s law, reciprocal space, Fourier transform, Friedel and Bijvoet pairs, Ewald’s sphere, Patterson function). 3. Protein crystallography – data collection Mounting the crystal (mother liquor - soaking, cryoprotection). X-ray sources (diffractometers, synchrotrons). Collecting the reflections (detectors - resolution, intensities). 4. Protein crystallography – data analysis Crystal symmetry, unit cell and image scaling (space groups, indexing – Miller indexes, scaling, merging). Initial phasing (phase problem, amplitude vs. phase, structure factor, molecular replacement, anomalous x-ray scattering SAD or MAD, multiple isomorphous replacement). Model building and phase refinement (B-factors, R-factors). Structure deposition (PDB database). 5. Circular dichroism – secondary and tertiary protein structure determination Physical principles (circular polarization of light, interaction of circularly polarized light with matter). Applications to biological molecules (far-UV spectrum – secondary structure, near-UV spectrum – tertiary structure, visible UV – metal-protein interactions). Experimental limitations (synchrotrons vs. home-sources, oxygen absorption, concentration, additives). PRACTICAL EXERCISES 1. Protein crystallography - crystallization Preparation of protein samples and crystallization solutions. Usage of different crystallization techniques. Take up with high through-put techniques and UV-imaging. 2. Protein crystallography – data collection Data collection of an exemplary protein crystal (from practical exercise – protein crystallization) on a home-source diffractometer. Test the crystal diffraction quality. Optimize the exposure time, detector distance. Take diffraction patterns in 0 and 90 degrees and optimize the start and the end of the data collection (MOSFLM program). Collect the complete series of diffraction patterns for protein crystal. 3. Protein crystallography – data analysis Analyze one complete data-set of a protein crystal. Indexing of the data-set (MOSFLM or XDS program) and scaling-merging of the data-set (CCP4 program package, SCALA). Solving the phase problem with a usage of molecular replacement and structure refinement (CCP4 program package and Coot). 4. Circular dichroism spectroscopy Preparation of protein samples and standard substance for CD measurements. Defining the standard curves for secondary structures of the proteins with the help of standard substance. Analyzing the CD spectra of the protein sample. Collecting the CD spectra of the protein sample in near UV region and defining the regions for aromatic residues.
Literatura
    doporučená literatura
  • PECORA R. Dynamic Light Scattering: Applications of Photon Correlation Spectroscopy, Springer, 1985
  • Bergfors, T. M. Protein Crystallization: Second Edition, Oxford University Press, 2009.
  • Fundamentals of crystallography. Edited by Carmelo Giacovazzo. 2nd ed. Oxford: Oxford University Press, 2002, xix, 825 s. ISBN 0-19-850957-X. info
  • MAREK, Jaromír a Zdeněk TRÁVNÍČEK. Monokrystalová rentgenová strukturní analýza. 1. vyd. Olomouc: Univerzita Palackého v Olomouci, 2002, 169 s. ISBN 8024405512. info
  • Circular dichroism and the conformational analysis of biomolecules. Edited by Gerald D. Fasman. New York: Plenum Press, 1996, ix, 738 s. ISBN 0-306-45142-5. info
Výukové metody
Intensive course covering lectures, class discussion, and practical exercises
Metody hodnocení
in-term assessment, final written test
Vyučovací jazyk
Angličtina
Další komentáře
Předmět je dovoleno ukončit i mimo zkouškové období.
Předmět je vyučován každoročně.
Výuka probíhá blokově.
Předmět je zařazen také v obdobích podzim 2011 - akreditace, podzim 2015, podzim 2016, podzim 2017, podzim 2018, podzim 2019, podzim 2020, podzim 2021, podzim 2022, podzim 2023, podzim 2024.