X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a 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, as well as their chemical bonds, their crystallographic disorder, and various other information.

1. X-ray crystallography
Analytical technique in plant science
32167501
Dipti yadav
Roll no. – 18556051, Botany hons, Kalindi college, Delhi University
Image source - https://commons.wikimedia.org/wiki/File:Freezed_XRD.jpg
01 Your name
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2. Index
1
Introduction
2
History
3
Principle of crystallography
4
X-ray diffraction
5
Bragg’s Law
6
Application
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3. Introduction
• 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.
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4. History
• The English physicist Sir William Henry Bragg pioneered the determination of
crystal 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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5. X-ray crystallography
• X-ray crystallography (XRC) is the experimental science determining the atomic
and molecular structure of a crystal, in which the crystalline structure causes a
beam of incident X-rays to diffract into many specific directions.
• By measuring the angles and intensities of these diffracted beams,
a 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, as well as their chemical bonds,
their crystallographic disorder, and various other information.
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6. Principle 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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7. 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
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8. Bragg’s Law
Bragg’s Law was introduced by Sir W.H. Bragg and his son Sir W.L. Bragg.
The law states that when the x-ray is incident onto a crystal surface,
its angle of incidence θ, will reflect back with a same angle of scattering θ.
And, when the path difference, dd is equal to a whole number, nn, of wavelength,
a constructive interference will occur. Consider a single crystal with aligned plane
of lattice points separated by a distance d. Monochromatic X-rays A, B, and
C are incident upon the crystal at an angle θ. They reflect off atoms X, Y, or Z.
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9. Bragg’s Law
nλ = 2d sinƟ
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10. Bragg’s Law
• Crystals are regular arrays of atoms, and X-rays can be considered waves of
electromagnetic radiation. Atoms scatter X-ray waves, primarily through the atoms'
electrons.
• X-ray striking an electron produces secondary spherical waves emanating from the
electron. This phenomenon is known as elastic scattering, and the electron is known as
the scatterer. A regular array of scatterers produces a regular array of spherical waves.
• Although these waves cancel one another out in most directions through destructive
interference, they add constructively in a few specific directions, determined by Bragg's law
• X-rays are used to produce the diffraction pattern because their wavelength λ is typically
the same order of magnitude (1–100 angstroms) as the spacing d between planes in the
crystal. In principle, any wave impinging on a regular array of scatterers
produces diffraction, as predicted first by Francesco Maria Grimaldi in 1665.
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11. 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 mand charge q is:
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12. • 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
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13. T
ypesofX-raydevice 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 suchas a rotating anode
tube or a strong synchrotron x-ray source for analyzing the protein crystals.
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14. First step
The process begins by crystallizing a protein of interest. 4 critical stepsare
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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15. 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 thex-rays
going through the crystal and the rest being scattered in variousdirections.
The crystal is rotated so that the x-rays are able to hit the protein fromall
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 thebasic
experimental data of the analysis.
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16. 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 filmto
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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17. 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
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18. 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.
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19. Applications of X-ray Crystallography
X-Ray Analysis of MilkPowder
This technique has also been used in study of milk powder. Most work hasbeen 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.
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20. References:
• Hickman, A. B. and Davies, D. R. (2001). Principles of macromolecular X-ray crystallography.
• Current Protocols in Protein Science, Chapter 17, Unit 17.3. New York: Wiley Interscience.
• Miao, J., Ishikawa, T., Shen, Q. and Earnest, T. (2008). Extending X-ray crystallography to allow the imaging of
noncrystalline materials, cells, and single protein complexes. Annual Reviews in Physical Chemistry, 59, 387–410.
• Mueller, M., Jenni, S. and Ban, N. (2007). Strategies for crystallization and structure determination of very large
macromolecular assemblies. Current Opinion in Structural Biology, 17, 572–579.
• Wlodawer, A., Minor, W., Dauter, Z. and Jaskolski, M. (2008). Protein crystallography for noncrystallographers, or
how to get the best (but not more) from published macromolecular structures. FEBS Journal, 275, 1–21.
• WEBSITES
http://www.colorado.edu/physics/2000/xray/index.html
http://www.physics.upenn.edu/~heiney/talks/hires/hires.html
http://www.matter.org.uk/diffraction/x-ray/default.htm
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21. Thank you
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