Daniel Böhringer: Catalogue data in Spring Semester 2019
|Name||Dr. Daniel Böhringer|
ScopeM - Cryo Electron Microscopy
ETH Zürich, HPM C 51
|Telephone||+41 44 633 39 85|
|551-0436-00L||Cryo-electron Microscopic Studies of Ribosomal Complexes with Biomedically Important Viral mRNAs |
Number of participants limited to 15.
The enrolment is done by the D-BIOL study administration.
|6 credits||7G||N. Ban, D. Böhringer, M. A. Leibundgut|
|Abstract||Some viral mRNAs, such as from Hepatitis C virus, hijack cellular translational machinery by binding directly to the ribosome and circumventing the need for cellular initiation factors. They accomplish this through structured elements within the mRNAs called internal ribosome entry sites (IRESs). Participants of this course will visualize ribosomes in complex with viral IRESs at high resolution.|
|Objective||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.|
|Content||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. Viral messenger RNAs are often produced by their own machinery, however, and need to be incorporated into the host translation machinery without the usual processing and therefore many viruses have developed strategies to circumvent the need for initiation factors. They accomplish this through highly structured elements within their RNA called internal ribosome entry sites (IRESs) that are able to initiate translation without the normal signals. Some viral IRESs, such as the IRESs from polio-virus or HIV, require most of the normal eIFs and even additional proteins. Others, such as the hepatitis C virus IRES, are able to bind directly to the ribosome and need only a few of the normal initiation factors. Within the Ban lab, we have studied, and continue to investigate, medically relevant viral IRESs. The course will involve producing, and attempting to determine the structures of, IRESs that have yet to have had their ribosome-bound structures resolved.|
A variety of purification techniques, including preparative gel electrophoresis 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.
|Lecture notes||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).
|Prerequisites / Notice||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.
|551-1412-00L||Molecular and Structural Biology IV: Visualizing Macromolecules by X-Ray Crystallography and EM||4 credits||2V||N. Ban, D. Böhringer, T. Ishikawa, M. A. Leibundgut, K. Locher, M. Pilhofer, K. Wüthrich, further lecturers|
|Abstract||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.|
|Objective||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.|
|Content||February 22 Lecture 1 Prof. Dr. Kurt Wüthrich |
History of Structural Molecular Biology
March 1 Lecture 2 Prof. Dr. Kaspar Locher
X-ray diffraction from macromolecular crystals
March 8 Lecture 3 Prof. Dr. Kaspar Locher
Data collection and statistics, phasing methods
March 15 Lecture 4 Prof. Dr. Nenad Ban
Crystal symmetry and space groups
March 22 Lecture 5 Ban Lab
Practical session with X-ray data processing
March 29 Lecture 6 Prof. Dr. Takashi Ishikawa
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)
April 5 Lecture 7 Dr. Daniel Boehringer
Single particle analysis, including principle (projection matching, random conical tilt, angular reconstitution)
April 12 Lecture 8 Ban Lab
Practical session including specimen preparation (cryo, negative stain, visit to ScopeM
May 3 Lecture 9
Prof. Dr. M. Pilhofer
Tomography I, including basics and subtomogram averaging
May 10 Lecture 10 Ban Lab
Practical session with example initial EM data processing
May 17 Lecture 11 Prof. Dr. Martin Pilhofer
Practical session (including recent techniques, including cryo-FIB)
May 24 Lecture 12 Prof. Dr. Nenad Ban
EM and X-ray structure building, refinement, validation and interpretation
May 31 Lecture 13 Ban Lab
Practical session with model building and refinemen