Marc André Leibundgut: Katalogdaten im Frühjahrssemester 2021
|Herr Dr. Marc André Leibundgut
Inst. f. Molekularbiol.u.Biophysik
ETH Zürich, HPK H 5
|+41 44 633 31 48
|Cryo-Electron Microscopic Studies of Ribosomal Complexes with Biomedically Important Viral Proteins
Number of participants limited to 15.
The enrolment is done by the D-BIOL study administration.
General safety regulations for all block courses:
-Whenever possible the distance rules have to be respected
-All students have to wear masks throughout the course. Please keep reserve masks ready. Surgical masks (IIR) or medical grade masks (FFP2) without a valve are permitted. Community masks (fabric masks) are not allowed.
-The installation and activation of the Swiss Covid-App is highly encouraged
-Any additional rules for individual courses have to be respected
-Students showing any COVID-19 symptoms are not allowed to enter ETH buildings and have to inform the course responsible
|N. Ban, D. Böhringer, M. A. Leibundgut, T. Lenarcic
|Many viruses have evolved specialized mechanisms to hijack the host gene expression machinery and employ cellular resources to regulate viral life cycle. They accomplish this through producing non-structural proteins that can, among other things, inhibit host protein synthesis. Participants of this course will visualize ribosomes in complex with a non-structural viral protein at high resolution.
|The goal of the course is to acquire the most important techniques and methods for the purification and structural characterisation of macromolecular complexes by transmission electron microscopy. The emphasis of the course is on the special practical requirements for the application of these techniques on macromolecular structures in the MDa range.
|Protein synthesis is a very energy intensive process that can consume over half the total metabolism of a cell. In eukaryotes, translation is therefore tightly regulated at the stage of initiation. Regulatory processes are much more complex at this step than in prokaryotes and a large number of RNA modification processes and translation initiation factors are required to ensure faithful initiation, elongation and termination of translation. However, several viruses may interfere with host translation by affecting the initiation step or by modifying the activity of key initiation factors to ensure an efficient translation of viral mRNA. Amongst such viruses is also SARS-CoV-2, which infects a large variety of vertebrate species. On entering host cells, the viral genomic RNA is translated by the cellular protein synthesis machinery to produce a set of non-structural proteins, which by inhibiting host translation render cell conditions favorable for viral production. Within the Ban lab, we have studied, and continue to investigate, medically relevant viral proteins. This course will involve producing and attempting to determine the structure of a non-structural viral protein in a ribosome-bound form.
A variety of purification techniques, including affinity chromatography and ultracentrifugation, will be used during the purification of macromolecular complexes. Purified assemblies will be then investigated functionally. Students will then characterise their samples structurally through transmission electron cryo-microscopy (cryo-EM), including sample preparation, microscopy, data evaluation and the calculation of densities. Finally, students will learn how to build and refine molecular models into parts of the calculated cryo-EM density. The participants will be working on a closed project related to current research within the laboratory and throughout the course the practical work will be accompanied by brief theoretical introductions. The principal aim of the course is to strengthen the skills required to independently conduct meaningful biophysical and biochemical experiments and to provide an early introduction into the structural characterisation of cellular macromolecular assemblies.
|A script will be distributed at the beginning of the course that will cover the experiments to be performed, provide references to the relevant literature and suggest points for further consideration for interested students.
A basic overview is provided within the references below. Further reading and citations shall be detailed in the course script.
- A. Fersht, Structure and mechanism in protein science, Freeman, 1999 (Chapters 1 and 6).
- M. van Heel et al., Single-particle electron cryo microscopy: towards atomic resolution, Quart. Rev. Biophys. (33), 307-369 (2000).
|Voraussetzungen / Besonderes
|The course will be held in English. Students should have either completed courses:
551-0307-00L Biomolecular Structure and Mechanism I: Protein Structure and Function
551-0307-01L Biomolecular Structure and Mechanism II: Large Cellular Machines
or equivalent courses covering the structure and function of biological macromolecules.
|Molecular and Structural Biology IV: Visualizing Macromolecules by X-Ray Crystallography and EM
|N. Ban, D. Böhringer, T. Ishikawa, M. A. Leibundgut, K. Locher, M. Pilhofer, K. Wüthrich, weitere Dozierende
|This course provides an in-depth discussion of two main methods to determine the 3D structures of macromolecules and complexes at high resolution: X-ray crystallography and cryo-electron microscopy. Both techniques result in electron density maps that are interpreted by atomic models.
|Students will obtain the theoretical background to understand structure determination techniques employed in X-ray crystallography and electron microscopy, including diffraction theory, crystal growth and analysis, reciprocal space calculations, interpretation of electron density, structure building and refinement as well as validation. The course will also provide an introduction into the use of cryo-electron tomography to visualize complex cellular substructures at sub-nanometer resolutions, effectively bridging the resolution gap between optical microscopy and single particle cryo-electron microscopy. Lectures will be complemented with practical sessions where students will have a chance to gain hands on experience with sample preparation, data processing and structure building and refinement.
|- History of Structural Molecular Biology
- X-ray diffraction from macromolecular crystals
- Data collection and statistics, phasing methods
- Crystal symmetry and space groups
- X-ray data processing
- Principle of cryo-EM for biological macromolecules I, including hardware of TEM and detectors, image formation principle (phase contrast, spherical aberration, CTF), 3D reconstruction (central-section theorem, backprojection, missing information)
- Single particle analysis, including principle (projection matching, random conical tilt, angular reconstitution)
- Tomography I, including basics and subtomogram averaging
- Tomography - recent techniques, including cryo-FIB
- EM specimen preparation (cryo, negative stain), initial EM data processing
- EM and X-ray structure building, refinement, validation and interpretation
- Model building and refinement