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X-ray Crystallography Maviya Naznin Koly South East University

X- ray Crystallograpy

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Page 1: X- ray Crystallograpy

X-ray Crystallography

Maviya Naznin KolySouth East University

Page 2: X- ray Crystallograpy

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.

Introduction

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HISTORYThe 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.

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Principles of X-ray Crystallography 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).

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X-ray diffractionX-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

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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.

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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:

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Instrument ComponentsGenerally a typical x-ray diffraction contain below parts:

1. Detector2. X-ray source3. Crystal on the end of mounting needle4. Liquid nitrogen steam to keep crystal cold5. Movable mount to rotate crystal

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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.

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ProcedureFirst 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.

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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.

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Third StepAn 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.

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Applications of X-ray CrystallographyHIV-

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

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Applications of X-ray CrystallographyApplications 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.   

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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.

Dif ferent ia t ion 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.