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:

  1. Crystals irradiated with X-rays.

  2. Electrons scatter X-rays.

  3. Recombined scattered waves collected on a detector.

  4. 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.