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.