[3DEM] Three-dimensional Electron Microscopy

[3DEM] Three-dimensional Electron Microscopy

Protein structure determination using Three-Dimensional Electron Microscopy is one of the three core experimental techniques in Structural Biology. This module will provide an introduction to practical and theoretical aspects of the technique. No similar module is offered at this level anywhere in South Africa.

Lecturer

Associate Professor Trevor Sewell, Programme Co-ordinator, and Director of the Electron Microscope Unit (an inter-faculty and regional resource centre), also has experience in computer-based education, and is actively engaged in research in the field of structural biology. In addition to his co-ordinating role, Prof Sewell will present lectures on the determination of the three-dimensional structures of cells using tomography and single particle methods for the determination of macromolecular structure. He will also present workshops explaining some of the major achievements of structural biology studies on large structures including the ribosome.

Main Outcomes

The ability to:

  • prepare negative stain and cryo specimens of proteins, collect data using an electron microscope and reconstruct proteins in three dimensions using the single particle technique and iterative real space helical refinement.
  • understand the theory of protein structure determination using three-dimensional Electron Microscopy;
  • understand and use an electron microscope;
  • demonstrate an understanding of image formation in the electron microscope as it is applicable to biological objects;
  • demonstrate a knowledge of the component subsystems of the electron microscope and how they work;
  • be able to record images of biological material by cryo-electron microscopy and negative stain techniques;
  • demonstrate an understanding of the process of three dimensional reconstruction by the "single particle method";
  • produce scripts for image processing using SPIDER;
  • reconstruct both a helical and non-helical object using SPIDER and other programmes as necessary.

This module descriptor document also can be downloaded as an MS-Word document.

Syllabus

  • Image Formation in the Transmission Electon Microscope.
  • Subsystems of the Transmission Electon Microscope.
  • Training in operation of preparative equipment and the TEM.
  • Image processing and 3D-reconstruction.

Main Content

  • Sample preparation
  • Design and operation of the electron microscope
  • Particle selection and classification
  • Assignment of projection angles
  • Three-dimensional reconstruction
  • Display and model building
  • Combination with crystallographic data
Home Department: Molecular and Cell Biology (UCT)
Credit Value: 15 Credits
Duration: 6 Weeks
Module Type: P
Level: 8
Prerequisites: Computational and Mathematical Primer (STB 705),
Structural Cell Biology (SBT 705),
Protein Expression and Purification Primer,
Structural Bioinformatics and Molecular Modelling,
Protein Crystallography
Co-requisites: None
Prohibited combinations: None
Learning time breakdown (hours):
Contact with lecturer/tutor: 30
Assignments & tasks: 40
Tests & examinations: 0
Practicals: 40
Selfstudy: 40
Total Learning Time 150
Methods of Student Assessment: Students will be required to submit a practical write up and complete two assignments demonstrating mastery of the single particle technique and iterative real space helical refinement.Assignments - 100%

Assessment

Students will be required to submit a practical write up and complete two assignments demonstrating mastery of the single particle technique and iterative real space helical refinement. Students should adequately describe their methods, results and conclusions. Correct SPIDER scripts should be included in the writeup as appendices and any innovative methods should be indicated.

Tutorials

References

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Crowther, RA, DJ de Rosier, and A. Klug. 1970. "The reconstruction of a three-dimensional structure from projections and its application to electron microscopy." Proceedings of the Royal Society of London. A. Mathematical and Physical Sciences, 317 (1530): 319-340.

Crowther, RA and A Klug. 1975. "Structural analysis of macromolecular assemblies by image reconstruction from electron micrographs." Annual Review of Biochemistry, 44 (1): 161-182.

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