X-Ray Crystallography is a technique used to determine the atomic and molecular structure of a crystal, in which the crystalline atoms cause a beam of incident X-rays to diffract into many specific directions.

1. X-RAY Crystallography
By Mehwish Nawaz

2. Introduction
• X-ray Crystallography is a scientific method of
determining the precise positions/arrangements
of atoms in a crystal where beams of X-ray
strikes a crystal and causes the beam of light to
diffract into many specific directions.

3. Electromagnetic Spectrum
X-Ray

4. Cont…
• A technique used to determine the atomic and
molecular structure of a crystal, in which the crystalline
atoms cause a beam of incident X-rays to diffract into
many specific directions.
• The atomic planes of a crystal cause an incident beam
of X- rays to interfere with one another as they leave
the crystal. The phenomenon is called X-ray diffraction.
A stream of X-rays directed at a crystal diffract and
scatter as they encounter atoms.
• The scattered rays interfere with each other and
produce spots of different intensities that can be
recorded on film.

5. X-Ray Diffraction Pattern
• X-ray diffraction or X-ray crystallography uses
the uniformity of light diffraction of crystals 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

6. Principle
• The principle is based on principle of diffraction
• The crystal is made to strike against x-ray beam.
• Due to striking the atoms present in crystal
diffracts the x-ray beam into different direction.
• The angle and intensity of this diffraction rays is
analog to spatial arrangement of atom in crystal.
• By studying these angle, the 3D structure of any
crystal can be determine.

7. Instrumentation
• X-ray Tube
The source of X rays
• Incident-beam optics
Condition the X- ray beam before it hits the sample
• The goniometer
The platform that holds and moves the sample, and
detector as well as the sample & sample holder
• Receiving-side optics
• Condition the X- ray beam after it has encountered the
sample
• Detector
Count the number of X rays scattered by the sample

8. Instrumentation

9. X-Ray Diffraction

10. Bragg’s Law
• Bragg’s 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, d is equal to a
whole number, n, of wavelength, a
constructive interference will occur.

11. Bragg’s Law

12. Bragg’s Law
• Bragg’s Law
nλ = 2d sinƟ
where:
• λ is the wavelength of the x-ray,
• d is the spacing of the crystal layers (path
difference),
• θ is the incident angle (the angle between
incident ray and the scatter plane), and
• n is an integer

13. Bragg’s Law

14. Types of interference
• Constructive
interference
• Troughs and crests align
= Amplitudes of waves
add together
• Destructive
interference
• Troughsandcrests are
not aligned and their
amplitudes cancel each
other out

15. Partial Interference
• Most common
• Only slightly unaligned
leads to complex pattern
of high and low
amplitudes

16. X-Ray Diffraction Pattern
• X-ray diffraction uses the uniformity of light
diffraction of crystals 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

17. Electron density map
• 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

18. Steps of XRD
• Protein purification
• Protein crystallization
• Shooting X-Rays on Protein crystals
• Data collection
• Structure Solution (Phasing)
• Structure determination

19. 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.

20. Protein Sample for Crystallization
• Pure and homogenous (identified by SDS-PAGE, Mass Spec. etc.)
• Properly folded Stable for at least few days in its crystallization condition (dynamic
light scattering)
Conditions that Effect Crystallization
• pH (buffer) Detergent
• Protein Concentration Temperature
• Pressure Precipitant
• Size and shape of the drops
• Salt (Sodium Chloride, Ammonium Chloride etc.)

21. Reason to crystallize a protein?
• Researchers crystallize an atom or molecule,
because the precise position of each atom in a
molecule can only be determined if the
molecule is crystallized.
• If the molecule or atom is not in a crystallized
form, the X-rays will diffract unpredictably and
the data retrieved will be too difficult if not
impossible to understand.

22. Second Step
• X-rays are generated by bombarding electrons
on an metallic anode.
• 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 (Goniometer).

23. Production of X-Rays

24. 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.

25. Overview of XRD

26. Structure Solution (Phasing)
• A typical diffraction
pattern from a protein
crystal
• The 3D structure
obtained above is the
electron density map
of the crystal.
GOAL= From Diffraction Data to Electron
Density

27. Fourier Transform
• A Fourier transform is done to extract the
frequency-domain spectrum from the raw time-
domain spectrum.
• Because we have different waves of X-rays
superimposed on one another during diffraction,
it is difficult to isolate the contribution of each
diffraction event to determine the lattice
structure. Therefore a mathematical tool known
as the Fourier transform is used.

28. Resolution needed for the
interpretation of electron density map
• Resolution needs to be taken into the
following account:
• A resolution of 5Å - 10Å can reveal the
structure of polypeptide chains,
• 3Å - 4Å of groups of atoms,
• 1Å - 1.5Å of individual atoms.

29. Advantages
• Least expensive, the most convenient.
• Widely used method to determine crystal
structures.
• X-Rays are not absorbed very much by air, so
the sample need not be in an evacuated
chamber.

30. Disadvantages
• X-Rays do not interact very strongly with
lighter elements.
• The intensity is 108 times less than that of
electron diffraction.

31. Applications
• To identify crystalline phases and orientation
• To determine structural properties
• To measure thickness of thin films and multi-
layers
• To determine atomic arrangement
• Detection limits: ~3% in a two phase mixture;
can be~0.1% with synchrotron radiation