Advances in Structural Biology (E05X0A)

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Algemeen

At the end of the course the student must understand the biophysical foundations of structural biology and the role of structural biology in modern biomedical research. (S)he should know the principles of the main experimental methods used (crystallography, SAXS, NMR, electron microscopy etc), and be ready to apply these methods in his/her own research.


Examenvorm

Type : Continuous assessment without exam during the examination period Description of evaluation : Paper/Project, Participation during contact hours Type of questions : Open questions Learning material : Course material, Computer, Reference work No 2nd examination opportunity

The evaluation will be based on the following components: (a) successful participation in questions / problem solving sessions (oral or written), (70%) and (b) active participation in all discussions (30%). You must be present for all lectures unless there is a valid reason, to be communicated with the tutor. If you fail to do so, you get zero grade for the course.

Bestanden

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Sjabloon:Advances in Structural Biology (E05X0A)/bestanden


Examenvragen

(Klik hier om examenvragen toe te voegen.) Structural Biology Research Track: Questions for the written test on 18.12.2014 (‘etc…’ means ‘continue the list’ – show your knowledge!) 1. What is the research area of structural biology? 2. Which types of host organisms can be used for recombinant expression of proteins? What are the advantages and disadvantages of each host system? 3. How is a plasmid to express a protein in E. coli culture constructed? Which elements does it contain? 4. A protein obtained from a tissue or through recombinant expression is typically highly contaminated with other proteins, DNA, lipids etc. Describe the methods that can be used to further purify such a protein sample. 5. How does denaturing electrophoresis of proteins (such as SDS-PAGE) work, and what data can be obtained using this method? 6. Size exclusion chromatography (also known as gel filtration) can be used either for purification or as an analytical method. Explain how it works. What sort of data can be obtained using analytical gel filtration? 7. Describe the principles and possible setups for protein crystallization. 8. What is called ‘resolution’ (of an atomic structure, electron microscopy envelope, etc…)? 9. What resolution is achievable using various methods of structural biology (X-ray crystallography, electon microscopy, NMR, etc…)? 10. Once a macromolecular crystal is obtained, atomic structure can be determined based on measuring the X-ray diffraction from the crystal. Describe the main steps of this procedure. 11. What is the ‘phase problem’ of crystallography? How can it be solved? 12. What is the meaning of the ‘crystallographic R-factor’? Provide a formula if you can. 13. A refined atomic model of a macromolecule (such as determined using crystallography, NMR, etc) must have ‘good geometry’. What does this mean? 14. How does small-angle X-ray scattering (SAXS) on solution work, and what data can be obtained? 15. Describe the principles of determining macromolecular structures using nuclear magnetic resonance (NMR). What are the advantages and limitations of NMR compared to X-ray crystallography? 16. How does an electron microscope work, and what data can be obtained (explain different possible techniques)? 17. How much detail can be resolved using (a) X-rays and (b) electron beams (in an electron microscope) and why? 18. How does an atomic force microscope work, and what data can be obtained? 19. How does analytical ultracentrifugation work, and what data can be obtained? 20. How does protein calorimetry (such as differential scanning calorimetry and isothermal titration calorimetry) work, and what data can be obtained? 21. How does a mass spectrometer work? How accurately can it determine the molecular masses? 22. Which methods can be used to measure the characteristic size (e.g. in terms of twice the hydrodynamic radius) of a macromolecule? 23. Which methods can be used to measure the mass of a macromolecule? 24. Which methods can be used to assess the shape of a macromolecule? 25. Two proteins X and Y make a stable complex in solution. Which methods can be used to evaluate the contact interface between X and Y (i.e. which residues of either protein are involved, etc…) 26. Only few atomic structures of membrane proteins have been determined until now, compared to those of the soluble proteins. What are the extra challenges of membrane proteins? 27. Describe the main principles and procedures of ‘structure-based drug design’. 28. What is the definition of the dissociation constant KD, with respect to a drug molecule binding to its target protein? How can KD be measured/estimated? Any reference material (Internet, notes, books,…) is not allowed. Supplementing your answer with drawings (schemas, etc) is allowed.


Structural Biology Research Track: Questions for the written test on 18.12.2014 (‘etc…’ means ‘continue the list’ – show your knowledge!)

1. What is the research area of structural biology?

2. Which types of host organisms can be used for recombinant expression of proteins? What are the advantages and disadvantages of each host system?

3. How is a plasmid to express a protein in E. coli culture constructed? Which elements does it contain?

4. A protein obtained from a tissue or through recombinant expression is typically highly contaminated with other proteins, DNA, lipids etc. Describe the methods that can be used to further purify such a protein sample.

5. How does denaturing electrophoresis of proteins (such as SDS-PAGE) work, and what data can be obtained using this method?

6. Size exclusion chromatography (also known as gel filtration) can be used either for purification or as an analytical method. Explain how it works. What sort of data can be obtained using analytical gel filtration?

7. Describe the principles and possible setups for protein crystallization.

8. What is called ‘resolution’ (of an atomic structure, electron microscopy envelope, etc…)?

9. What resolution is achievable using various methods of structural biology (X-ray crystallography, electon microscopy, NMR, etc…)?

10. Once a macromolecular crystal is obtained, atomic structure can be determined based on measuring the X-ray diffraction from the crystal. Describe the main steps of this procedure.

11. What is the ‘phase problem’ of crystallography? How can it be solved?

12. What is the meaning of the ‘crystallographic R-factor’? Provide a formula if you can.

13. A refined atomic model of a macromolecule (such as determined using crystallography, NMR, etc) must have ‘good geometry’. What does this mean?

14. How does small-angle X-ray scattering (SAXS) on solution work, and what data can be obtained?

15. Describe the principles of determining macromolecular structures using nuclear magnetic resonance (NMR). What are the advantages and limitations of NMR compared to X-ray crystallography?

16. How does an electron microscope work, and what data can be obtained (explain different possible techniques)?

17. How much detail can be resolved using (a) X-rays and (b) electron beams (in an electron microscope) and why?

18. How does an atomic force microscope work, and what data can be obtained?

19. How does analytical ultracentrifugation work, and what data can be obtained?

20. How does protein calorimetry (such as differential scanning calorimetry and isothermal titration calorimetry) work, and what data can be obtained?

21. How does a mass spectrometer work? How accurately can it determine the molecular masses?

22. Which methods can be used to measure the characteristic size (e.g. in terms of twice the hydrodynamic radius) of a macromolecule?

23. Which methods can be used to measure the mass of a macromolecule?

24. Which methods can be used to assess the shape of a macromolecule?

25. Two proteins X and Y make a stable complex in solution. Which methods can be used to evaluate the contact interface between X and Y (i.e. which residues of either protein are involved, etc…)

26. Only few atomic structures of membrane proteins have been determined until now, compared to those of the soluble proteins. What are the extra challenges of membrane proteins?

27. Describe the main principles and procedures of ‘structure-based drug design’.

28. What is the definition of the dissociation constant KD, with respect to a drug molecule binding to its target protein? How can KD be measured/estimated?

Any reference material (Internet, notes, books,…) is not allowed. Supplementing your answer with drawings (schemas, etc) is allowed.