Experiment Title: Protein Structure Determination by X-ray Crystallography
Introduction:
X-ray crystallography is a powerful technique used in biophysical chemistry to determine the three-dimensional structure of proteins. This experiment demonstrates the principles of X-ray crystallography and how it can be used to study the structure of a protein. It involves growing high-quality protein crystals, exposing them to X-rays, and then interpreting the resulting diffraction pattern to build a 3D model of the protein.
Key Procedures:
1. Protein Purification and Crystallization:
- Purify the protein of interest using techniques such as chromatography (e.g., size exclusion, ion exchange, affinity chromatography). Ensure high purity to obtain well-ordered crystals.
- Optimize crystallization conditions by varying parameters such as protein concentration, pH, temperature, and the presence of precipitants (e.g., polyethylene glycol, ammonium sulfate). This often involves screening a range of conditions using techniques like hanging drop vapor diffusion.
- Monitor crystal growth over time, aiming for well-formed crystals of sufficient size and quality for X-ray diffraction.
2. Data Collection:
- Carefully mount a single, well-diffracting protein crystal on a goniometer head (a device that allows precise orientation of the crystal).
- Expose the crystal to a monochromatic X-ray beam (typically from a synchrotron source for high-intensity and quality data).
- Collect diffraction data using a detector, recording the intensity and angle of scattered X-rays. This data is a series of spots representing the diffracted X-rays from the crystal lattice.
3. Data Processing and Structure Determination:
- Process the raw diffraction data to correct for various factors (e.g., background noise, detector response). Software packages are used for this step.
- Index the diffraction spots to assign Miller indices, representing the crystal lattice planes.
- Calculate the electron density map using phasing techniques (e.g., molecular replacement, isomorphous replacement, anomalous scattering). Phasing is crucial to solve the 'phase problem' and obtain interpretable electron density.
- Build a molecular model of the protein into the electron density map using molecular modeling software. This is an iterative process of fitting amino acid residues into the electron density, refining the model to best fit the data.
4. Model Refinement and Validation:
- Refine the protein model to optimize its fit to the electron density and to minimize steric clashes and other inconsistencies.
- Validate the model using various criteria such as R-factor, R-free, stereochemistry checks, and Ramachandran plot analysis. These assess the quality and accuracy of the resulting structure.
Significance:
- X-ray crystallography provides a high-resolution view of protein structure, revealing crucial details about its conformation, active sites, and interactions with other molecules.
- Understanding protein structure is vital for comprehending its function, mechanism of action, and role in biological processes.
- Structural information enables the rational design of drugs and therapies targeting specific proteins, as well as the engineering of proteins with improved properties.
- X-ray crystallography has been instrumental in advancing our understanding of various fields, such as enzymology, immunology, and structural biology.