X-ray crystallography is a technique used to determine the atomic and molecular structure of crystals. X-rays are directed at a crystal and the diffraction pattern produced is analyzed to reveal the crystal structure. This information can be used to construct a 3D electron density map and determine atomic positions. Some important applications of X-ray crystallography include determining the structures of proteins, viruses like HIV, and using this information to develop new drugs.

1. X-ray Crystallography
Maviya Naznin Koly
South East University

2. X-ray crystallography is a powerful technique for visualizing the structure of protein.
It is a tool used for identifying the atomic and molecular structure of a crystal.
In crystallography the crystalline atoms cause a beam of incident X-rays to diffract
into many specific directions.
Then crystallographer can produce a three-dimensional picture of the density of
electrons within the crystal.
From this electron density, the mean positions of the atoms in the crystal can be
determined.
X-ray crystallography can locate every atom in a zeolite, an aluminosilicate.

4. The English physicist Sir William Henry Bragg pioneered the determination of
crustal structure by X-ray diffraction methods
X-ray crystallography is a complex field that has been associated with several of
science’s major breakthroughs in the 20th century
Using X-ray crystal data, Dr. James Watson and Dr. Francis Crick were able to
determine the helix structure of DNA in 1953.
In 1998 Dr. Peter Kim, a scientist, was able to determine the structure of a key
protein responsible for the HIV infection process.

5. Ray diffraction by crystals is a reflection of the periodicity of crystal architecture,
so that imperfection in the crystal lattice usually results in poor diffraction
properties.
A crystal can be described with the aid of grid or lattice, defined by three axis and
angles between them.
Along each axis a point will be repeated as distances referred to as the unit cell
constants, labeled a, b and c.
Within the crystalline lattice, infinite sets of regularly spaced planes can be drawn
through lattice points.
These pinlanes can be considered as the source of diffraction and are
designated by a set of three numbers called the Miller indices(hkl).

6. X-ray diffraction
X-ray crystallography uses the uniformity of light diffraction of crytals to
determine the structure of molecule or atom
Then X-ray beam is used to hit the crystallized molecule.
The electron surrounding the molecule diffract as the X-rays hit them.
This forms a pattern. This type of pattern is known as X-ray diffraction pattern

7. Bragg’s Law
nλ = 2d sinƟ
Here d is the spacing between diffracting planes, Ɵ is the incident angle, n is any
integar, and λ is the wavelength of the beam.
These specific directions appear as spots on diffraction pattern called reflections.
Thus X-ray diffraction results from an electromagnetic wave impinging on a
regular array of scatters.

8. Thomson scattering
The X-ray scattering is determined by the density of electrons within the crystal.
Since the energy of an X-ray is much greater than that of a valence electron, the
scattering may be modeled as Thomson scattering, the interaction of an
electromagnetic ray with a free electron
The intensity of Thomson scattering for one particle with mass m and
charge q is:

9. Generally a typical x-ray diffraction contain below parts:
1. Detector
2. X-ray source
3. Crystal on the end of mounting needle
4. Liquid nitrogen steam to keep crystal cold
5. Movable mount to rotate crystal

10. Types of X-ray device used
Elecrons are responsible for the diffraction and intensity in crystallography
Electrons they scatter x-rays weaker than heavy elements.
Knowing this, protein crystallographers use high intensity x-ray sources such as
a rotating anode tube or a strong synchrotron x-ray source for analyzing the
protein crystals.

11. First step
The process begins by crystallizing a protein of interest. 4 critical steps are
taken to achieve protein crystallization:
Purify the protein. Determine the purity of the protein and if not pure (usually
>99%), then must undergo further purification.
Protein must be precipitated by dissolving it in an appropriate solvent(water-
buffer soln. w/ organic salt such as 2-methyl-2,4-pentanediol).
The solution has to be brought to supersaturation by adding a salt to the
concentrated solution of the protein.
Let the actual crystals grow. Since nuclei crystals are formed this will lead to
obtaining actual crystal growth.

12. Second Step
X-rays are generated and directed toward the crystallized protein
Then, the x-rays are shot at the protein crystal resulting in some of the x-rays
going through the crystal and the rest being scattered in various directions.
The crystal is rotated so that the x-rays are able to hit the protein from all
sides and angles.
The pattern on the emulsion due to scattering reveals much information
about the structure of the protein.
The intensities of the spots and their positions are thus are the basic
experimental data of the analysis.

13. Third Step
An electron density map is created based on the measured intensities of the
diffraction pattern on the film
A Fourier Transform can be applied to the intensities on the film to
reconstruct the electron density distribution of the crystal
The mapping gives a three-dimensional representation of the electron
densities observed through the x-ray crystallography
When interpreting the electron density map, resolution needs to be taken into
account
A resolution of 5Å - 10Å can reveal the structure of polypeptide chains, 3Å -
4Å of groups of atoms, and 1Å - 1.5Å of individual atoms.

14. Applications of X-ray Crystallography
HIV-
Scientists also determined the X-ray crystallographic structure of HIV
protease, a viral enzyme critical in HIV’s life cycle, in 1989.
Pharmaceutical scientists hoped that by blocking this enzyme, they could
prevent the virus from spreading in the body.
By feeding the structural information into a computer modeling program,
they could use the model structure as a reference to determine the types of
molecules that might block the enzyme.
Arthritis-
To create an effective painkiller in case of arthritis that doesn’t cause
ulcers, scientists realized they needed to develop new medicines that shut
down COX-2 but not COX-1.
Through structural biology, they could see exactly why Celebrex plugs up
COX-2 but not COX-1

15. Applications of X-ray Crystallography
Applications of X-Ray Crystallography in Dairy Science
X-ray crystallography technique has been a widely used tool for elucidation of
compounds present in milk and other types of information obtained through
structure function relationship.
Stewart has shown that even solutions tend to assume an orderly arrangement
of groups within the solution.
Hence, liquid milk should, and does show some type of arrangement.
The mineral constituent and lactose are the only true crystalline constituents in
dairy products that can be analyzed by X-ray.
Analysis of Milk Stones
X-ray diffraction technique has also been applied for analysing the chemical
composition of milk stones. Since each chemical compound gives a definite
pattern on a photographic film according to atomic arrangement, X-rays can be
used for qualitative chemical analysis as well as structural analysis.

16. Applications of X-ray Crystallography
X-Ray Analysis of Milk Powder
This technique has also been used in study of milk powder. Most work has been
confined to determine the effect of different milk powdering processes upon
structural group spacings within the milk proteins.
Differentiation of Sugar
Since each crystalline compound gives a definite pattern according to the
atomic arrangement, the identification and the differentiation of the common
sugars (sucrose, dextrose and lactose) is made simple by X-rays
In case of new material
X-ray crystallography is still the chief method for characterizing the atomic
structure of new materials and in discerning materials that appear similar by
other experiments.