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Lecturer(s)
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Kutá Smatanová Ivana, prof. Mgr. Ph.D.
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Course content
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Lectures: From protein expression and purification to protein crystallization Introduction to protein crystallization Principles of protein crystallization Preparation of protein samples for crystallization experiments Protein crystallization screening Conventional crystallization methods and their modifications Crystallization under oil Advanced crystallization techniques Counter diffusion methods for protein crystallization and screening Lipidic cubic phase crystallization Microseeding with automatic systems Preparation of micro- and nanocrystals for free-electron-laser and synchrotron radiation sources Membrane protein crystallization Interpretation of the crystallization drop results Crystallization and crystallographic analysis in a microfluidic chip Tips and tricks for protein crystal manipulation Crystal mounting and freezing Large volume crystal growth in restricted geometry for neutron crystallography Subjects of the practical exercises within the course: 1. Conventional techniques and their modifications, crystallization of own proteins using commercial screening kits - using standard crystallization techniques, modification of conventional techniques, using oil, commercial screens (material: commercially available proteins + students' own proteins) 2. Crystallization of membrane proteins in lipidic mesophases 3. Microfluidic system crystallization in nanovolumes 4. Automates for crystallization crystallization robot demonstration, microbatch method 5. Protein crystallization using the GCB - GAME and other methods using capillaries, preparation a large and small diameter capillary counter diffusion for neutron diffraction 6. Observation of crystal growth / Seeding check of crystallization set-ups, application of seeding techniques to improve crystal quality 7. The secret life of your crystallization drop 8. Crystal mounting and freezing crystal mounting using capillaries, mounting to nylon loops and freezing in liquid nitrogen, crystal storage and transport
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Learning activities and teaching methods
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Monologic (reading, lecture, briefing), Dialogic (discussion, interview, brainstorming), Work with text (with textbook, with book), Demonstration, Projection, Laboratory
- Class attendance
- 65 hours per semester
- Preparation for credit
- 10 hours per semester
- Preparation for exam
- 10 hours per semester
- Preparation for classes
- 10 hours per semester
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Learning outcomes
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X-ray crystallography is known to be one of the most important techniques for the study of proteins, nucleic acids and their complexes, as well as viruses and supramolecular complexes based on atomic resolution. Crystallization of biomacromolecules is an empirical method, which is based on finding individual conditions and parameters for crystal growth and optimization. Since 2004, the Crystallization Course has been organized every two years as a FEBS Practical Crystallization Course "Advanced Methods in Macromolecular Crystallisation". The course is lectured by leading world experts in protein crystallography. In the morning cycles of lectures, the students will learn methods of isolation, purification and crystallization of proteins, with standard procedures, but also with the latest trends. In the afternoon, in the framework of practical exercises, students will try everything they have learned at the lectures. During the lab exercises, students will grow the crystals of model and/or own proteins, plus test all new crystallization methods and become familiar with crystal measurement on a diffractometer.
Graduates will became familiar with methods and techniques of crystallisation of important biomolecular compounds like proteins and nucleic acids. They will be able to find by own individual conditions and parameters for growing crystals and their optimalization using standard, advanced and alternative crystallisation techniques, moreover they will know how to test character of crystals and prepare crystals for diffraction measurements on the X-ray sources (diffractometers and synchrotrons) for a final determination of macromolecular structure.
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Prerequisites
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Completion of a general and organic chemistry and biochemistry courses and laboratory work. Since teaching is in English, English is also an important requirement.
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Assessment methods and criteria
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Student performance assessment, Combined exam, Development of laboratory protocols
Requirement for successful completion of the course is the active participation of the student in practical exercises. The final exam will take the form of a written test and a subsequent oral interview with a final mark in the form of a mark.
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Recommended literature
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A. Ducruix, R. Giege. Crystallization of NA and Proteins, A Practical Approach. Oxford University Press, NY, USA, 2006. ISBN 0-19-963678-8.
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I. Kutá Smatanová. Crystallization techniques in protein crystallography. 2008.
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J. Drenth. Principles of Protein X-ray Crystallography. Springer Verlag Publishing, USA, 2007. ISBN 978-0-387-33334-2.
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T.M. Bergfors. Protein Crystallization Techniques, Strategies and Tips. International University Line, CA USA, 1999. ISBN 0-9636817-5-3.
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