Data collection and processing are critical steps in X-ray crystallography, as they determine the quality and resolution of the final structure.
During data collection, the crystal is exposed to a beam of X-rays, and the diffracted X-rays are detected and recorded. The crystal is rotated in small increments, and the diffraction pattern is recorded at each orientation. The diffraction pattern is usually collected on a detector, such as an image plate, CCD detector or a pixel array detector.
The diffraction pattern collected from the crystal contains information about the positions and intensities of the diffracted spots, which are used to calculate the structure factors. The structure factors are a set of numbers that describe the amplitude and phase of the diffracted X-rays, and are essential for solving the crystal structure.
Once the diffraction data is collected, it must be processed to extract the structure factors. The raw diffraction data typically undergoes several processing steps to improve the signal-to-noise ratio and to correct for errors and artifacts.
The first step in data processing is indexing, which involves determining the orientation of the crystal and the unit cell parameters. The unit cell is the repeating unit of the crystal lattice, and its determination is critical for calculating the structure factors accurately.
After indexing, the diffraction spots are integrated, which involves measuring the intensities of the diffracted X-rays at each position on the detector. This step can be challenging, particularly for weak diffraction spots, and requires careful background subtraction and correction for systematic errors.
Finally, the processed data is scaled, which involves normalizing the intensities of the diffraction spots to account for differences in the X-ray flux and crystal quality. The scaling step is essential for obtaining accurate structure factors and determining the resolution of the diffraction data.
Overall, data collection and processing are critical steps in X-ray crystallography, as they determine the quality and resolution of the final structure. The accuracy and precision of the structure factors and the diffraction data are crucial for obtaining a high-quality structure that can provide insights into the molecular function and mechanism of biological molecules.