Distance Learning: Techniques in Structural Molecular Biology (TSMB)

Electron Microscopy (EM) is a technique that is complementary to X-ray crystallography as it enables the study of non-crystalline, very large or transient macromolecular structures.

• Historical background
• Macromolecular electron microscopy

TEM and SEM
Image formation
EM and X-Ray Diffraction Compared
The Fourier Transform
Phases

• EM of Macromolecular Assmblies
• Sample Preparation

Support Films
Negative Stain
Cryo-EM

• Data Collection

Transferring the Sample
Film versus CCD
Digitizing Images

• Data Processing

Problems
The Contrast Transfer Function
Noise
Picking and Sorting Particles
2-D Projections

• 3-D Reconstruction
• Refinement
• Gro-EL: An Example of 3-D Single Particle Reconstruction
• Electron Microscopy of 2-D Crystals

Growth of 2-D Crystals
Data Collection
Image Processing

• Helical Arrays
• Electron Microscopy of Viruses
• Tomography

Objectives

To introduce the students to the principles of and techniques used in studying protein structures using electron microscopy methods. To illustrate how images are obtained and explain the importance of resolution. To compare the different EM techniques available to structural biologists. At the end of this section, students should:

• Understand the different EM methods used in structural biology, and when each one is used
• Understand image processing as it applies to reconstructing structures from EM results at a very basic level
• Know how symmetry averaging is used in 2-D and 3-D electron crystallography
• Understand the importance of validation
• Appreciate the current limitations of EM as a tool in structural biology