10064 24-25 8.3 Analytical techniques III Protein Structure determination(1)

Page 1: Overview of Analytical Techniques

• Course Reference: LSC-10064

• Date: 19/01/2025

• Topic: Analytical Techniques III - Protein Structure Determination.

• Institution: Keele University, School of Life Sciences.

Page 2: Analytical Techniques in Protein Structure Determination

• Chromatography: A separation technique based on size, charge, hydrophobicity, and specificity.

  • Types include:

    • Gel filtration

    • Ion exchange

    • Affinity chromatography

    • High-Performance Liquid Chromatography (HPLC)

    • Reversed Phase

    • Gas Chromatography (GC)

• Electrophoresis: Separation of macromolecules like DNA, RNA, and proteins by size and/or charge.

  • Techniques include:

    • SDS PAGE

    • Isoelectric focusing

    • 2D gel electrophoresis

• Spectroscopy: Determines structure by measuring absorption and transmission of electromagnetic radiation.

  • Techniques include:

    • Infrared spectroscopy

    • UV-visible spectroscopy

    • Nuclear Magnetic Resonance (NMR)

• Mass Spectrometry: Structural characterization through fragmentation and mass measurement.

  • Methods include:

    • MALDI

    • ESI

    • MS-MS

    • GC-MS

• X-ray Crystallography: Interpreting scattering patterns from crystalline molecular arrays to determine structure.

Page 3: Methods for Determining Protein Structure

• 1. X-ray Crystallography: Atomic resolution for enzymes, antibodies, receptors, viruses.

• 2. Nuclear Magnetic Resonance (NMR): High-resolution details mainly for small proteins.

• 3. Electron Microscopy (EM): Evolving to provide atomic-level details for larger proteins.

Page 4: Protein Structure Determination Statistics

• Experimental Method Structures (Data as of October 2024):

  • X-ray: 188,747

  • Electron Microscopy: 23,203

  • NMR: 14,400

  • Other methods (e.g., Multi Method, Neutron): 319

  • Total: 226,707 structures recorded.

Page 5: Comparative Methods - PX vs Cryo-EM

• Analysis of X-ray crystallography (PX) and Cryo-electron microscopy in structure determination.

Page 6: Logarithmic Scale Visualization

• Visual representation of X-ray crystallography and EM techniques results using a logarithmic scale.

Page 7: Nobel Prize in Chemistry 2017

• Awarded for Cryo-electron microscopy.

• Recipients: Richard Henderson, Jacques Dubochet, Joachim Frank.

Page 8: X-ray Crystallography Process

• Process Steps:

  1. Crystallize proteins.

  2. Expose crystals to X-ray.

  3. Detect scattered X-rays.

  4. Analyze patterns for atomic arrangement.

Page 9: T-Cell Receptor Structural Representation

• Diagrams of MHC I and MHC II T-cell receptors, referencing structural biology resources.

Page 10: Overview of Proteins Studied

• Includes notable proteins like C-reactive protein and Surfactant protein D.

Page 11-12: General Principles of X-ray Crystallography

• Key Principles:

  • Interaction of X-rays with electron density in crystals.

  • Diffraction patterns reveal atomic locations and quantities.

Page 13: Molecular Integrity in Crystals vs In Vivo

• Maintained integrity in crystals.

• Crystals consist of 50-75% solvent.

• Enzymatic activity retained.

Page 14: Requirement for X-ray Scattering Patterns

• Need for significant quantities of protein molecules (approx. 10^12) arranged in a crystal.

Page 15-20: Overview of Protein Crystal Characteristics

• Typical size and composition of protein crystals (0.1-0.2 mm, 50-70% solvent).

• Techniques to optimize crystal growth.

Page 21-24: Crystal Growth Process

• Steps in molecular aggregation and crystallization processes.

• Conditions for crystallization adjustment.

Page 25-27: Crystallization Methods

• Sitting Drop and Hanging Drop methods for crystallization via vapor diffusion.

Page 28: Microcrystal Example and Screening Methods

• Example conditions for microcrystals and screening methods using sparse matrices.

Page 29-30: Crystallization of Membrane Proteins

• Importance of solubility in crystallizing membrane-bound proteins.

Page 31-32: Case Studies in Membrane Protein Crystallization

• Example: Bacteriorhodopsin crystallization process and conditions.

Page 33-34: Challenges in Crystal Mounting

• Crystals' fragility necessitates careful mounting in a maintained aqueous environment.

Page 35-36: Cryocrystallography and Preservation

• Techniques to preserve protein integrity during crystallization and X-ray exposure.

Page 37: Importance of Order in X-ray Patterns

• Reflection of crystal order through diffraction patterns.

Page 40: Overview of Structure Determination Methodologies

• Emphasis on measurement accuracy and AI’s growing role in structure prediction.

Page 41: Comparison of Techniques

• Techniques compared:

  • X-ray Crystallography

  • NMR

  • Cryo-Electron Microscopy

Page 42-44: Nobel Prizes in Chemistry

• 1988 Prize: Johann Deisenhofer, Robert Huber, Hartmut Michel for structure determination of biomolecules.

• 2017 Prize: for development of cryo-electron microscopy.