S1004 Methods for structural characterization of biomolecules

Faculty of Science
Autumn 2018
Extent and Intensity
1/1/0. 2 credit(s) (plus extra credits for completion). Recommended Type of Completion: zk (examination). Other types of completion: k (colloquium).
Mgr. Josef Houser, Ph.D. (lecturer)
prof. RNDr. Michaela Wimmerová, Ph.D. (lecturer)
Guaranteed by
prof. RNDr. Michaela Wimmerová, Ph.D.
National Centre for Biomolecular Research - Faculty of Science
Supplier department: National Centre for Biomolecular Research - Faculty of Science
Prerequisites (in Czech)
Course Enrolment Limitations
The course is offered to students of any study field.
The capacity limit for the course is 10 student(s).
Current registration and enrolment status: enrolled: 0/10, only registered: 1/10, only registered with preference (fields directly associated with the programme): 0/10
Course objectives
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
Learning outcomes
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
  • 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.
    recommended literature
  • 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 and 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
Teaching methods
Intensive course covering lectures, class discussion, and practical exercises
Assessment methods
in-term assessment, final written test
Language of instruction
Further comments (probably available only in Czech)
The course can also be completed outside the examination period.
The course is taught annually.
The course is taught: in blocks.
Note related to how often the course is taught: October 23-26, 2017.
The course is also listed under the following terms Autumn 2011 - acreditation, Autumn 2014, Autumn 2015, Autumn 2016, autumn 2017, Autumn 2019.
  • Enrolment Statistics (Autumn 2018, recent)
  • Permalink: https://is.muni.cz/course/sci/autumn2018/S1004