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Protein Structure Determination by X-ray Crystallography

🧬 Protein Structure Determination by X-ray Crystallography

📌 Purpose & Context

  • Goal: Determine atomic structure of proteins.

  • Applications:

    • Drug design (active site info).

    • Understanding binding mechanisms.

🔍 X-ray Crystallography

✅ Advantages

  • High-resolution structures.

  • Can trap intermediates (via flash freezing).

  • Suitable for small & large proteins.

  • Rapid (esp. with synchrotron sources).

❌ Limitations

  • Typically done at −173 °C (not physiological).

  • Static, average structure.

  • Hydrogens often not visible.

  • Membrane proteins are hard to crystallize.

🔄 Alternative Methods

🔹 NMR (Nuclear Magnetic Resonance)

  • Good for small, dynamic proteins.

  • Difficult sample prep.

  • Uses radio waves.

🔹 Electron Microscopy (Cryo-EM)

  • Great for large complexes.

  • Low protein concentrations required.

  • Needs cryogenic samples.

🔬 The Crystal

  • Crystal: Ordered 3D array of molecules.

  • Unit cell: Smallest repeating unit.

  • 3 axes (a, b, c) and 3 angles (α, β, γ).

🔧 Why Crystals?

  • Bragg’s Law: Constructive interference when path difference = nλ.

  • Crystals amplify X-ray signal due to uniform atomic planes.

🧪 Crystallization

Process:

  1. Supersaturate protein solution.

  2. Add precipitant → nucleation → crystal growth.

Variables:

  • pH, buffer, salt type/concentration, temperature, protein concentration.

Techniques:

  • Hanging drop: Good visibility/control.

  • Sitting drop: Automation/high throughput.

  • Vapour diffusion: Equilibration increases protein/precipitant concentration slowly.

Challenges:

  • No universal rules—trial and error.

  • Requires pure, concentrated, stable protein.

  • Not all proteins crystallize.

❄ Cryocooling Crystals

  • Flash freeze with cryoprotectant (e.g., 25% glycerol).

  • Reduces radiation damage.

  • Traps enzyme intermediates and oxidation-sensitive states.

🔬 Data Collection

🔹 X-ray Sources

  • Laboratory: Fixed wavelength (1.54 Å), low brightness, slow (1–2 days).

  • Synchrotron: Tunable (0.6–1.6 Å), fast (minutes), higher brightness.

🔹 XFELs (Free Electron Lasers)

  • SFX (Serial Femtosecond Crystallography):

    • Streams microcrystals.

    • Captures ultrafast events.

📷 Diffraction

  • X-rays diffract off electron clouds.

  • Data recorded as reflections (spots) on detector.

  • Rotate crystal → build 3D dataset.

  • Pattern is in reciprocal space (closer spots = wider atomic spacing).

📏 Resolution

  • Measured in Ångströms (1 Å = 0.1 nm).

  • Lower Å = higher resolution.

  • X-rays detect electrons (hydrogens poorly visualized).

⚠ Phase Problem

  • X-ray measures amplitude, not phase.

  • Phase must be estimated to build model.

Solutions:

  • Molecular Replacement: Needs model with >30% identity.

  • Experimental Phasing:

    • MAD (Multi-wavelength Anomalous Diffraction)

    • SAD (Single-wavelength)

    • MIR (Multiple Isomorphous Replacement)

🛠 Model Building & Validation

Tools:

  • Fit atomic model into electron density map.

  • Validation:

    • Bond lengths/angles.

    • Ramachandran plot (φ, ψ angles of amino acids).

    • Side-chain rotamers.

    • Difference maps.

    • B-factor (temperature factor) checks.

🧪 Application Example: SARS-CoV-2 Spike Protein

  • Spike protein bound to ACE2 receptor.

  • Stabilized by 4 disulfide bonds (oxidized cysteines).

  • Crystallography helped determine structure for vaccine design.