Around 100 years ago William Henry Bragg and his son William Lawrence Bragg used Sodium Chloride crystal to pass x ray beams through it that created a beautiful geometric pattern on a photographic paper placed behind it.
Being inspired by the experiment of Rosalind Franklin in 1953 producing images (also known as photograph 51) of crystal DNA from X ray, Watson & Crick described the double helical structure of DNA by ‘Diffraction pattern analysis’.
X-rays are electromagnetic radiation with wavelengths between about 0.02 Å and 100 Å. Because X-rays have wavelengths similar to the size of atoms, they are used to explore within crystals. Today it is scientifically established that X rays hitting these particulates obstacles interpret the atoms inside a crystal lattice to give a structure of regular geometric patterns.
X ray diffraction analysis is mainly diffraction pattern that is observed when the shower of electrons after diffracting the X ray pass through a crystal unit. Electron density map can be obtained from these diffraction patterns. These maps show contour lines of electron density. Since electrons more or less surround atoms uniformly, it is possible to determine where atoms are located. To get a three-dimensional picture, the crystal is rotated with a view to getting a three-dimensional picture in together with a detector connected to a computer producing two-dimensional electron density maps for each rotation.
Unfortunately, there is no known way to focus x-rays with a lens, unlikely with visible lights. Until then it is necessary to use crystals to diffract x-rays and create a diffraction pattern which can be interpreted mathematically by a computer. This turns the computer into a virtual lens to help us look at the structure of a molecule. Crystals have a repeated cell unit within them and so the x-ray diffraction from one unit cell would not be significant. Fortunately, a good crystal diffraction pattern is observed which is amplified enough to produce a 3D picture in a detector due to multiple cell units within a crystal.
There are various methods of growing protein crystals for x ray diffraction analysis:
Vapor Diffusion – (Hanging Drop Method)
The most common ways of crystal growth where a drop of protein solution is suspended over a reservoir containing buffer and precipitant. Water diffuses from the drop to the solution leaving the drop with optimal crystal growth conditions.
Crystals are grown by keeping a saturated protein solution put in a sealed container.
By lowering the saturation over time, diffusion of proteins into the oil is made happen by putting a drop of protein solution in inert oil and let the crystal grow.
By growing a crystal in a highly saturated solution and then placing it in a less saturated one to let the crystal growth happen.
A few crystals are grown, then crushed, and put into a final solution that combines them into a few nice crystals. This involves quite a bit of experimentation with solutions’ concentrations to get the desired number of crystals.
Free interface diffusion
A container has levels of varying saturation. Crystals form initially in the highly saturated part, but as the solution mixes, it eventually only supports crystal growth.
Similar to the previous, but with a semipermeable membrane separating the layers.
How does X ray crystallography work?
- X-ray beams are shot through a crystal of the atom keeping the crystal mounted to a Goniometer to keep it in place during the operation
- X-Ray after being diffracted through the crystal lattice leads the beam in a pattern based on their structure.
- A diffraction pattern is then finally observed.
Advantages of x ray crystallography technique:
- The nondestructive nature of X ray makes it suitable to characterize metals.
- Knowing the wavelength and diffraction angle and with the use of Bragg’s law, information on the crystalline condition of the sample, and thus phase proportions; texture and degree of preferred orientation can be calculated.
- Plastic strain and particle size can be interpreted using peak width.
Limitations of X ray crystallography technique:
- Requires a crystal with the shortest side of 0.2 mm
- Crystallization may require conditions that may not be physiological.
Flaws of Crystallization:
- Disorder in Unit Cell
- Vibrations of molecules
- Distortion in crystallization
- Diffraction peaks are very crowded for complex macromolecules and are difficult to separate.
- Heavy atom substitution works for molecules larger than 600 atoms, so a gap is present for molecules in the intermediate size range.
Applications of X ray crystallographic technique:
- It is used to study materials such as salts, metals as well as various biological molecules etc. that can form crystal lattice structure.
- To determine the electron density, the mean positions of the atoms in the crystal, their chemical bonds, their disorders etc.
- To detect the size of atoms, and types of chemical bonds, and the atomic scale differences among various materials, especially minerals and alloys. The method also revealed the structure and function of many biological molecules, including vitamins, drugs, proteins and nucleic acids such as DNA
- To identify the atomic structure of new materials that may seem similar in other experiments
- X ray crystal structures can also account for unusual electronic or elastic properties of a material and also serve as the basis for designing pharmaceuticals against diseases.
The specificity of the protein’s active sites and binding sites is completely dependent on the protein’s precise conformation. X-ray crystallography can reveal the precise three-dimensional positions of most atoms in a protein molecule because x-rays and covalent bonds have a similar wavelength, and therefore currently provides the best visualization of protein structure. At present, this technique is helping the researchers to elucidate the folding of secondary protein residual structures depending on various external factors.
Rafat Shahriar Islam, is a graduate from Department of Pharmacy, East West University. He can be reached at firstname.lastname@example.org
Share your Idea or article by mailing at email@example.com with your name, institution and Photo.