FEDERAL UNIVERSITY,DUTSE
DEPARTMENT OF BIOCHEMISTRY
X-ray Crystallography
in Determination of Protein Structure
By
HADIZA MUHAMMAD ADAM
FSC/BCH/20/1018
X-RAY Crystallography
X-ray crystallography is an experimental technique used to determine the atomic and molecular
structure of crystals, including protein crystals. When an ordered crystal is exposed to x-rays, the
atoms’ electron clouds scatter the x-rays, creating a distinctive diffraction pattern. By analyzing the
intensities and angles of diffracted beams, researchers can compute a 3D electrons density map and
infer atomic positions and chemical bonds.
Principles behind x-ray crystallography is based on;
1. X-ray diffraction: X-rays are diffracted by the electrons in a crystal lattice, producing a diffraction
pattern.
2. Bragg’s Law: The diffraction pattern is determined by spacing between atoms in the crystal lattice,
as described by Bragg’s Law.
3. Reciprocal lattice: The diffraction pattern can be described in terms of the reciprocal lattice, which
is related to the real-space lattice of the crystal.
4. Fourier transform: The diffraction pattern is related to the electron density of the crystal through a
Fourier transform.
Key Steps in the Process
• Protein crystallization: Proteins are crystallized to obtain a ordered array of molecules (Mcpherson
and Gavira, 2014).
• X-ray diffraction: X-rays are diffracted by the crystals, producing a diffraction pattern (Dauter,
2006).
• Data processing: Diffraction data are processed to obtained structure factor amplitudes (Kabsch,
2010).
• Phasing: Phases are determined using methods such as molecular replacement or heavy atoms
derivatization (Taylor, 2003).
• Model building: An atomic model of the protein is built using the phased structure factor
amplitudes (Emsley et al., 2010).
• Refinement: The model is refined against the x-ray data to optimize its accuracy (Murshudov et
al., 2011).
Protein crystallization
The first requirement for protein structure determination by x-ray crystallography is the attainment
of protein crystals diffracting at high resolution. Protein crystallization is mainly a “trial and error”
procedure in which the protein is slowly precipitated from its solution. As a general rule, the purer
the protein, the better the chances to grow crystals. Growth of protein crystals starts from a super-
saturated solution of the macromolecules, and evolves towards a thermodynamically stable state in
which the protein is partitioned between a solid phase and the solution. The time required before the
equilibrium is reached has a great influence on the final result, which can go from an amorphous or
microcrystalline precipitate to large single crystals. The super-saturation conditions can be obtained
by addition of precipitating agents (salts, organic solvents and polyethylene glycol polymers) and/or
by modifying some of the internal parameters of the solution, like pH, temperature and protein
concentration. Since proteins are labile molecules, extreme conditions of precipitations, pH and
temperature should be avoided. Protein crystallization involves three main steps (Ducruix et al.,
1992).
1. Determination of protein degree of purity. If the protein is not at least 90-95% pure further
purification will have to be carried out to achieve crystallization.
Protein crystallization Cont’d
2. The protein should be dissolved in a suitable solvent from which it must be precipitated in
crystalline form. The solvent is usually a water buffer solution.
3. The solution is brought to super saturation. In this step small aggregates are formed, which are the
nuclei for crystal growth. Once nuclei have been formed, actual crystal growth begins.
