1. Introduction to Bioinformatics II
Lecture 13
X-Ray Crystallography
By Ms. Shumaila Azam

2. X-Ray Crystallography
A technique, using X rays, to determine the atomic structure of molecules that have been crystallized.
A technique for deducing molecular structure by aiming a beam of X rays at a crystal of the test compound and measuring the scatter of rays.
The technique requires good laboratory skills--without good crystals, good images will not be obtained.

3. Components
The three components needed to complete an X-ray crystallography analysis are
a protein crystal,
a source of x-rays and
a detector.

5. Technique
1st Step:
The process begins by crystallizing a protein of interest.
A protein must be precipitated out or extracted from a solution.
The rule of thumb here is to get as pure a protein as possible to grow lots of crystals
this allows for the crystals to have charged properties, and surface charged distribution for better scattering results

6. 4 critical steps are taken to achieve protein crystallization, they are:
Purify the protein. Determine the purity of the protein and if not pure
Must precipitate protein. Usually done so by dissolving the protein in an appropriate solvent(water-buffer soln. w/ organic salt such as 2-methyl-2,4-pentanediol. If protein is insoluble in water-buffer or water-organic buffer then a detergent such as sodium lauryl sulfate must be added).
The solution has to be brought to supersaturation. This is done by adding a salt to the concentrated solution of the protein, reducing its solubility and allowing the protein to form a highly organized crystal (this process is referred to as salting out).
Let the actual crystals grow.

7. 2nd Step
For the next step, x-rays are generated and directed toward the crystallized protein. X-rays can be generated in four different ways,
by bombarding a metal source with a beam of high-energy electrons,
by exposing a substance to a primary beam of X-rays to create a secondary beam of X-ray fluorescence,
from a radioactive decay process that generates X-rays
from a synchrotron (a cyclotron with an electric field at constant frequency) radiation source.

8. The patterns are a result of interference between the defracted x-rays governed by Bragg's Law:2dsinθ=n*λ ,
where d is the distance between two regions of electron density,θ is the angle of defraction, λ is the wavelength of the defracted x-ray and n is an integer.
If the angle of reflection satisfies the following condition:
Sinθ=(n*λ)/2d
the defracted x-rays will interfere constructively. Otherwise, destructive interference occurs.

9. Final step
The final step involves creating an electron density map based on the measured intensities of the diffraction pattern on the film.
the electron density distribution is illustrated as a series of parallel shapes and lines stacked on top of each other
The mapping gives a three-dimensional representation of the electron densities observed through the x-ray crystallography.

11. Myoglobin (a protein)
Crystal Crystallograph Structure

12. DNA Structure
The technique first developed by Maurice Wilkins and Rosalin Franklin and used by James Watson and Franics Crick to determine the double helix structure of DNA is known as X-ray crystallography.
X-rays are synchronized so that the crests and troughs are in identical locations.
These rays are then bounced off of the DNA molecule to form an image on photographic film.

13. DNA
Crystallograph Structure

14. Why X- Ray?
X-rays are used due to their low wavelength, between 0.05 angstrom and several hundred angstroms.
The wavelength of normal light is hundreds of times longer, making the offset caused by the molecule so small that it is of no use.
As the X-rays impact the DNA, they are reflected at different angles and at different times in their cycles (because the distance traveled is different for each wave).

15. However, when only one molecule is used, the image is very faint.
To enhance and amplify it the image, the molecules must be arranged in order, as in a crystal.
DNA is arranged in crystal form by turning it into a gel.
A small object can then be inserted into the mixture.
When twisted, the object will pull out a strand of several DNA molecules, all in order.
This strand can then undergo X-ray crystallography, and produce a much stronger image.