91Ó°ÊÓ

The two most difficult steps in X-ray crystallography are the crystallization of the protein and the determination of the electron density map from the diffraction data.

Crystallization of the protein is a challenging step because it requires obtaining a pure and homogeneous sample of the protein, growing a high-quality crystal, and finding the optimal conditions for crystallization. Many factors, such as protein concentration, temperature, and pH, can affect the crystal growth. Screening a vast number of crystallization conditions can be time-consuming and requires trial and error.

After obtaining a high-quality crystal, it is diffracted by X-ray beams to generate diffraction patterns. These diffraction patterns are then used to calculate the electron density map, which represents the distribution of electrons in the crystal. The phase problem is the main challenge in determining the electron density map: the diffraction data provides information about the amplitudes of the scattered waves, but not their phases. Since both amplitude and phase are required to calculate the electron density map, the missing phase information must be estimated. Various methods, such as molecular replacement, multiple isomorphous replacement (MIR), and single-wavelength anomalous diffraction (SAD), are used to estimate the phases. Each of these methods has its limitations and can be challenging to apply.