Study Notes on Transmission Electron Microscopy (TEM) - Dr. Mohinder Pal

Transmission Electron Microscopy (TEM) - Dr. Mohinder Pal

Overview

• Lecture Details: Led by Dr. Mohinder Pal, discussing Transmission Electron Microscopy (TEM) as a significant microscopy technique.

Recap of Techniques for Determining Protein Structures

• Nuclear Magnetic Resonance (NMR)

  • Resolution: Estimated 4-2 Å

  • Size Range: Usually < 30 kDa

  • Protein Quantity: Feasible < 100 kDa, requiring 10-100 mg of protein.

• X-ray Crystallography

  • Resolution: Typically 4-1 Å

  • Size Range: No upper limit

  • Protein Quantity: Requires 1-1000 mg.

• Electron Microscope (TEM)

  • Resolution: Typically 20-3 Å

  • Size Range: Usually > 500 kDa

  • Protein Quantity: Feasible > MDa, requiring only 0.1-1 mg of protein.

Learning Outcomes

• Define Transmission Electron Microscopy (TEM).

• Understand the evolution of TEM over the years.

• Explore the application of TEM in single particle cryo-electron microscopy (cryo-EM).

• Discuss sample preparation, focusing on protein purification.

• Evaluate sample preparation methods: Negative staining and cryo-EM techniques.

• Undertake structure determination methods.

• Examine three case studies involving TEM.

What is Transmission Electron Microscopy (TEM)?

• Definition: TEM is a microscopy technique where a beam of electrons is transmitted through the specimen to form an image.

• Specimen platform: Includes thick slices of cells or protein molecules dispersed on a circular grid.

• Camera Functionality: Captures images or records movies of the sample.

• Electron Source: Uses either a tungsten filament or a field emission gun.

Comparisons with Other Microscopy Techniques

• Fluorescence Microscopy: Limited resolution and imaging capabilities.

• Super-resolution Fluorescence Microscopy: Increased resolution but still not at the atomic level.

• X-ray Microscopy: Alternative imaging method with specific resolution

• Cryo Electron Microscopy (TEM):

  • Achieves atomic resolution (up to 1 nm).

  • Live cell imaging poses challenges due to resolution trade-offs.

Resolution in Light Microscopy vs. TEM

• Electron Microscope Resolution: c. 0.2 nm

• Optical Microscope Resolution: c. 200 nm

Power of Transmission Electron Microscopy

• CryoEM Images::

  • Displays projections of cellular edges at 1 micron.

  • Shows negative staining of tobacco mosaic virus (TMV) particles at 100 nm.

  • 3D reconstruction examples, such as double-layered rotavirus particles, with varying resolutions down to atomic levels.

• Applications: Connects cellular structures, molecular complexes, and secondary structures at high resolutions.

Resolution Principle

• Definition of Resolution: Closest spacing of two points that can still be resolved as separate entities.

• Abbe's Equation: $d = rac{ ext{N}}{2 n ext{sin}( heta)}$

  • Where:

  • $d$ = minimum separation of two points

  • $ext{N}$ = wavelength of light

  • $n$ = refractive index

  • $ext{sin}( heta)$ = numerical aperture (NA).

• Specimens must be imaged at wavelengths equal to or smaller than the target resolution.

Evolution in Transmission Electron Microscopy

• Historical Milestone: Invented by Ernst Ruska in 1931.

• Current Technology: Example of TITAN KRIOS equipped with 300 KeV and direct detectors.

Steps in Transmission Electron Microscopy

1. Sample Preparation: Includes protein expression and purification.

2. Negative Staining Analysis: Enhances visibility of samples.

3. Cryo Electron Microscopy Analysis: Allows for capture under frozen conditions.

4. Structure Determination: Involves the creation of 2D and 3D structural representations.

Sample Preparation: Protein Expression and Purification

1. Transformation: Introduction of DNA into host cells.

2. Selection: Utilize selective media to isolate transformed cells.

3. Cell Growth: Cultivation of cells to produce proteins.

4. Cell Lysis: Breaking the cells open to release proteins.

5. SDS-PAGE Analysis: Analyze protein size and purity.

6. Protein Chromatographies: Further purify using various chromatography techniques.

Sample Evaluation using Negative Staining

• Protocol Steps:

  • Adsorb a small drop of the protein sample onto a carbon support film.

  • Apply a heavy metal stain such as uranyl acetate.

  • Blot and air dry the sample before examination under a microscope.

• Purpose: Enhances contrast, allowing the shape of proteins like capsids to be discerned.

Sample Evaluation using Cryo-Electron Microscopy

• Cryo-EM Sample Preparation Protocol:

  • Use a grid for sample placement.

  • Utilize liquid ethane as a cryogen during plunge freezing of the sample.

• Outcome: Allows visualization of vitrified proteins without ice crystals interfering.

Structure Determination

• Process Overview:

  • Capture single particle images using TEM.

  • Use software like Relion/CryoSparc for alignment and class averaging.

  • Employ tools like Pymol, Coot, and Chimera for 3D refinement and mapping of side chains.

Precautions in 2D and 3D Model Generation

• Aim to mitigate noise in single particle cryo-EM as outlined by Henderson (2013).

Summary of Cryo-EM Workflow

• Flow Diagram:

  • Freeze sample and collect data.

  • Create 2D projections of particles on grids.

  • Perform particle alignment and averaging to develop a 3D model.

Case Studies

1. Cryo-EM Structure of R2TP Chaperone: Notable proteins include mTOR, ribosome components, and AAA-ATPases.

2. Cryo-EM Structure of ATP Synthase: Complex detail involving enzyme structures and ATP generation.

3. Cryo-Electron Tomography (cryo-ET): Used for obtaining 3D structures of ribosomes and nuclear pore complexes with resolution ranging from 1-4 nm.

Summary of Outcomes

• Students should have comprehended:

  • The definition and implication of Transmission Electron Microscopy (TEM).

  • The evolution and technological advancements in TEM.

  • The application of TEM in single particle cryo-electron microscopy (cryo-EM).

  • Insights into sample preparation and evaluation methods, focusing on protein structure determination techniques.