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:
Crystallize proteins.
Expose crystals to X-ray.
Detect scattered X-rays.
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.