10064 24-25 8.3 Analytical techniques III Protein Structure determination(1)
Page 1: Introduction to Analytical Techniques III
Title: Analytical Techniques III: Protein Structure Determination
Course Code: LSC-10064
Date: 19/01/2025
Institution: Keele University School of Life Sciences
Page 2: Overview of Analytical Techniques
Chromatography: Separation based on size, charge, hydrophobicity, composition, and specificity.
Types: Gel filtration, Ion exchange, Affinity, High-Performance Liquid Chromatography (HPLC), Reversed Phase, Gas Chromatography (GC).
Electrophoresis: Separates macromolecules (DNA, RNA, proteins) according to size and/or charge.
Techniques: SDS PAGE, Isoelectric focusing, 2D gel electrophoresis.
Spectroscopy: Structure determination by measuring the absorption and transmission of electromagnetic radiation.
Types: Infrared (IR), UV-visible, Nuclear Magnetic Resonance (NMR).
Mass Spectrometry: Structural characterization by fragmenting molecules to measure resulting masses.
Techniques: Matrix-Assisted Laser Desorption/Ionization (MALDI), Electrospray Ionization (ESI), MS-MS, and GC-MS.
X-ray Crystallography: Structure determination by interpreting scattering patterns from crystalline molecular arrays.
Page 3: Methods for Determining Protein Structure
High-resolution Electron Microscopy (EM): Provides atomic-level detail for large proteins and viruses.
X-ray Crystallography (PX or MX): Offers atomic-level details for proteins such as enzymes and antibodies.
Nuclear Magnetic Resonance (NMR): Provides high resolution for small proteins, but is less common for larger proteins.
Page 4: Experimental Method Statistics
Distribution of structures solved by various methods (as of October 2024):
Total Structures: 226,707
Methods include X-ray, Electron Microscopy, and NMR, detailing specific structural counts for each molecular type.
Page 5: X-ray Crystallography vs. Cryo-EM
Comparison of structure determination methods emphasizing the benefits of each and advances in techniques.
Page 6: Visualization of Experimental Structures
Use of logarithmic scales to illustrate the distribution of solved structures over time.
Page 7: Nobel Prize in Chemistry 2017
Awarded for the Development of Cryo-electron Microscopy.
Key Figures: Richard Henderson, Jacques Dubochet, and Joachim Frank.
Page 8: X-ray Crystallography Process
Structure determination follows:
Crystals irradiated with X-rays.
Electrons scatter X-rays.
Recombined scattered waves collected on a detector.
Analysis of scattered X-rays reveals atomic arrangement.
Page 9: T-cell Receptor Structure
Reference to figures pertaining to T-cell receptors and their roles in immune response.
Page 10: Examples of Analyzed Proteins
Mention of C-reactive protein and Surfactant protein D as examples of analyzed proteins.
Page 11: Recap of X-ray Crystallography
Emphasis on interpreting scattering patterns from crystalline molecular arrays.
Page 12: General Principles of X-ray Crystallography
Key principles include:
Interaction of radiation with matter (penetration, scattering, and potential damage).
Importance of X-ray beam and crystal relationship for data interpretation.
Page 13: Crystallization vs. In Vivo Structures
Integrity of proteins maintained during crystallization under physiological conditions (e.g., solvent adjustments).
Page 14: Process of X-ray Crystallography
Steps involved in obtaining interpretable scattering patterns with required conditions.
Page 15: Details on Protein Residues
Specific amino acid residues in C-reactive protein and their possible significance.
Page 16: Measurement Units in Crystallography
Summary of units used (Ångstroms) and their relevance to measurements in X-ray crystallography.
Page 17: Protein Crystal Formation
Explanation that billions of protein molecules need to be arranged in ordered patterns for analysis.
Page 18: Characteristics of Protein Crystals
Protein crystals are small, maintain biological activity, and exhibit significant solvent content.
Page 19: Conditions for Obtaining Protein Crystals
Factors affecting protein crystallization including pH, temperature, and solvent properties.
Page 20: Structure of Protein Crystals
Dynamics of crystallization focusing on regular array formation under suitable conditions.
Page 21: Stages of Crystal Growth
Description of stages in crystal growth as solubility decreases.
Page 22: 2D Array Formation during Crystallization
Details of molecular interactions forming two-dimensional arrays and unit cells.
Page 23: Crystallization Techniques
Methods to reach minimum solubility and form regular contacts between protein molecules.
Page 24: High-Throughput Crystallization Methods
Introduction of robotic methods for protein crystallization to enhance efficiency.
Page 25: Crystallization Techniques Using Vapor Diffusion (Sitting Drop)
Detailed setup description for sitting drop method in vapor diffusion crystallization.
Page 26: Crystallization Techniques Using Vapor Diffusion (Hanging Drop)
Detailed explanation of the hanging drop method for crystallization.
Page 27: Challenges in Protein Crystallization
Overview of challenges faced in achieving successful precipitation and crystallization.
Page 28: Example Crystallization Conditions
Reference to specific conditions that may lead to reproducible and well-ordered crystals.
Page 29: Membrane Proteins and Biochemistry
Importance of membrane proteins and challenges related to their crystallization.
Page 30: Crystallizing Membrane Proteins
Key strategies for obtaining soluble forms of membrane-bound proteins for crystallization.
Page 31: Example of Bacteriorhodopsin Crystallization
Specifics on how to make Bacteriorhodopsin soluble and crystalline.
Page 32: Nobel Prize in Chemistry 1988
Awarded for determining the 3D structure of the photosynthetic reaction center.
Page 33: Crystal Mounting Techniques
Overview of techniques used for mounting crystals for analysis.
Page 34: Maintaining Aqueous Environment for Crystal Mounting
Significance of maintaining an aqueous environment during crystal mounting to prevent damage.
Page 35: Cryocrystallography
Techniques used to maintain protein integrity and crystal lattice during X-ray exposure.
Page 36: Cryo-Preservation Techniques
Use of liquid nitrogen and related methods for preserving protein samples.
Page 37: X-ray Diffraction Patterns
Characteristics of diffraction patterns as reflections of order and symmetry within protein crystals.
Page 38: Recap of Protein Structure Determination
Summary of various techniques used in determining protein structure via X-ray crystallography.
Page 39: Structure Determination Add-ons
Reference to supplemental materials enhancing understanding of protein structures.
Page 40: Current Insights in Structure Determination
Discussion on the role of AI (e.g., AlphaFold) in structural prediction and its implications in research.
Page 41: Comparison of Techniques
Pros and cons of various analytical techniques such as X-ray crystallography, NMR, and Cryo-EM.
Page 42: Overview of Alphafold
Introduction to Alphafold by Google DeepMind and its significance in protein structure prediction.
Page 43: Nobel Prize in Chemistry 1988 Visuals
Images of laureates and their contributions to the determination of the photosynthetic reaction center.
Page 44: Nobel Prize in Chemistry 2017 Visuals
Visual representation of achievements in developing cryo-electron microscopy.