Macromolecular X-Ray Crystallography

Macromolecular X-Ray Crystallography

Introduction

  • Speaker: Dr. Jose Ortega-Roldan

  • Focus: The use of macromolecular X-ray crystallography in understanding molecular structures.

Research Areas

  • Topics in Research:

    • Membrane interactions

    • CLIC family of proteins

    • Related studies:

      • Varela & Hendry et al., JCS, 2022

      • Cassar et al., in preparation, 2023

      • Olotu, F., et al., CSBJ, 2023

    • Importance of Ca2+/Zn2+ interactions

    • Cl- ions significance

    • Low pH activation role in biological processes

  • Implications in Health:

    • Cancer

    • Cardiac conditions

Understanding T-Cell Receptor Activation

  • Key Components:

    • T-Cell Receptors (TCR)

    • H-2Db and 2m Peptide

  • Mechanism of Action:

    • Multiple peptides can bind to HLA, but not all induce an immune response.

X-Ray Crystallography Lectures

  • Topics Covered:

    • Diffraction theory

    • Crystallisation

    • Practical demonstrations of crystallography and analysis

    • Workshop for hands-on experience with data solving protein structures

Importance of X-Ray Crystallography

  • Purpose:

    • To determine the three-dimensional structure of molecules at atomic resolution.

    • Understanding structure leads to understanding biological function.

  • Molecular Insights:

    • Structures reveal processes such as enzyme activities, signaling pathways, and fundamental molecular processes like DNA translation to RNA to proteins.

X-Ray Fiber Diffraction

  • Visual Representation:

    • Blue bands depict sugar-phosphate chains.

    • Base pairs form horizontal connections; chains run in opposite directions (3' and 5').

    • Key Measurements:

    • Chain length: 2 nm - 3.4 nm

    • Distance between bases: 0.34 nm

    • Recognized Figures:

    • Francis Crick, James Watson, Maurice Wilkins, Rosalind Franklin

Nobel Prize Winners in Related Fields

  • Highlights of Contributions:

    • Max Perutz & John Kendrew (1962): Structure of hemoglobin and myoglobin

    • Frederick Sanger (1958): Sequence of insulin

    • Watson, Crick & Wilkins (1962): Molecular structure of nucleic acids

    • Dorothy Crowfoot Hodgkin (1964): Structures of significant biochemical substances via X-ray methods

    • Other notable winners include Deisenhofer, Huber & Michel (1988) for photosynthesis, and Paul Boyer & John Walker (1997) for ATP synthesis.

Drug Examples Targeting Molecular Structures

  • Novel Small Molecule Drugs:

    • Vemurafenib (Zelboraf/PLX4032):

    • Targets mutant BRAF V600E protein, frequently mutated in melanoma (50% cases) and in solid tumors (8% cases).

    • Imatinib (Glivec/Gleevec):

    • Effective in chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST) through inhibition of Bcr-Abl and c-Kit receptor tyrosine kinases.

Growth of Protein Crystallography

  • Data Representation:

    • A logarithmic scale indicating the number of structures analyzed over the years (1976 to 2023).

    • Growth patterns show increases in techniques like X-ray crystallography, electron microscopy (EM), and nuclear magnetic resonance (NMR).

Crystallization Fundamentals

  • Key Definitions:

    • Crystals: Ordered 3D arrays of molecules.

    • Asymmetric Unit: Smallest repeating unit within a crystal.

    • Unit Cell: Smallest volume element that is representative of the whole crystal (includes parameters such as axes a, b, c, and angles α, β, γ).

    • Lattice: An array of unit cell vertices.

Symmetry in Crystals

  • Concepts Covered:

    • Relation of left and right-handed forms through rotation and inversion.

    • Notion of symmetry including rotation, reflection, and inversion in three-dimensional space.

The Four Plane Lattices in Symmetry

  • Classification of Lattices:

    • Triclinic: No symmetry (primitive)

    • Monoclinic: One two-fold axis

    • Orthorhombic: Three orthogonal two-fold axes

    • Other crystalline symmetries include tetragonal, cubic, and hexagonal with specific arrangements of unit cells.

Electromagnetic Spectrum

  • Wavelength Ranges:

    • Various ranges from radio waves to gamma rays

    • X-ray wavelength range specifically useful in crystallography (0.1-0.2 nm)

  • Measurement Units:

    • 1 meter = 10² cm = 10³ mm = 10⁶ µm = 10⁹ nm = 10¹⁰ Å

Obtaining Images of Molecules via X-Rays

  • Principles:

    • X-rays must be shorter than the size of the object to obtain clear images.

    • Atoms are typically 0.15 nm apart, necessitating X-ray usage (e.g., λ = 0.154 nm for copper-target).

    • Hydrogen's position inferred from bond lengths and angles since it's often not directly seen in X-ray structures.

    • A crystal is required (containing 10¹³-10¹⁵ molecules) to produce measurable X-ray diffracted beams.

X-Ray Diffraction Experiment Setup

  • Components Required:

    • X-ray source (e.g., seal tube lamp), goniostat, detector, and collimator

    • Filters to ensure single wavelength (λ) X-ray radiation

    • Goniostat to allow full exposure of (hkl) planes to the X-ray beam.

Synchrotron Facilities for Advanced X-Ray Research

  • Functionality:

    • Charged particles are circulated near the speed of light, emitting electromagnetic radiation when changing directions.

  • Layout Elements in PROXIMA 1:

    • Robot sample changers, beam diagnostics, cryogenic cooling systems, monochromators.

Crystallographic Data Collection Techniques

  • Process Overview:

    • The crystal diffracts the X-ray source into discrete beams producing unique spots (reflections) used to determine molecular structures.

Bragg's Law

  • Formula:

    • nextλ=2dextsinhetan ext{λ} = 2d ext{sin} heta

    • Describes the relationship between wavelength (λ), distance between planes (d), and angle of diffraction (θ).

  • Resolution Insights:

    • Greater angles correspond to increased resolution (e.g., 2Å high resolution vs. 3Å low resolution).

Fourier Transform in X-Ray Crystallography

  • Purpose:

    • Establishes a relationship between an object and its diffraction pattern, crucial for generating electron density maps from diffraction data.

    • Converts diffraction patterns into volumetric representations of molecule structures.

  • Must know the position, intensity, and phase of reflections for accurate mapping (typically 10⁵ reflections).

Conclusion

  • Focus on Challenges in X-Ray Crystallography:

    • Obtaining adequate phase information remains a challenge, essential for deriving structural information from intensity measurements.

References and Sourcing

  • Mention of additional reading and online resources for further elaboration on crystallography techniques and applications, ensuring a comprehensive understanding of the field.