X-ray crystallography is a technique used to determine the atomic and molecular structure of crystals. It works by firing X-rays at a crystal and analyzing the diffracted rays. This allows researchers to construct a 3D model of the density of electrons within the crystal, revealing where atoms are located. Well-ordered protein crystals are required, as X-ray scattering from a single molecule would be too weak. Researchers grow crystals and collect diffraction data, which are then used to calculate atomic positions via Fourier transforms. This technique has determined over 85% of known protein structures and is invaluable for understanding functions at the molecular level.

1. X-ray Crystallography
By: Dr. Ashish C Patel
Assistant Professor
Vet College, AAU, Anand

2. • What is X-Ray Crystallography?
– A form of very high resolution microscopy.
– Enables us to visualize protein structures at the atomic level
– Enhances the understanding of protein function.
• What is the principle behind X-Ray Crystallography?
– It is based on the fact that X-rays are diffracted by crystals.

3. • X-ray crystallography is a method of determining the arrangement
of atoms within a crystal, in which a beam of X rays strikes a crystal
and causes the beam of light to spread into many specific
directions. From the angles and intensities of these diffracted
beams, a crystallographer can produce a three dimensional picture
of the density of electrons within the crystal.
• Because X-rays have wavelengths similar to the size of atoms, they
are useful to explore within crystals.
X RAY DIFFRACTION
• X-Ray Crystallography uses the uniformity of light diffraction of crystals to
determine the structure of a molecule or atom.
• Then they use an X-ray beam to “hit” the crystallized molecule. The
electrons surrounding the molecule diffract as the X-rays hit them. This
forms a pattern, this type of pattern is called the X-ray diffraction pattern.

4. Microscopy Wavelength Visualization
Light
Electron
X-Rays
300 nm
10 nm
0.1 nm or 1 Å
Individual cells and sub-cellular
organelles
Cellular architecture Shapes of
large protein molecules
Atomic detail of protein
Why X-Rays? Not Others?

5. X-ray
• In 1895, Wilhelm Conrad Roentgen, a German physicist
observed, while doing some experiments with the discharge
tube, that when cathode rays are allowed to fall on a metal
target called anticathode placed in their path, a new kind of
rays are produced. These radiations are called X-rays.

6. Properties of X-ray
• X-Rays are EM radiations having a wavelength between 10A to 0.01A
• In Free Space they travel in a straight line with a speed of 1,86,000
miles/sec (same as that of visible light)
• They are Invisible to Eye, Cannot be Heard or Smelt
• They cannot be Reflected, Refracted or Deflected by magnetic or
Electric Field
• They show properties of Interference, Diffraction and Refraction
similar to Visible light
• They Produce an Electric field at right angles to their path of
propagation
• It can penetrate liquids, solids and gases.
• X-rays interact with materials they penetrate and cause ionization
• X-Rays also have a germicidal or bactericidal effect
• X-rays are capable of producing an image on a photographic film

7. History
• 1895: William Roentgen discovers x-rays.
• 1912: Von Laue, Friedrich and Knipping publish “Interference
Effects with Roentgen Rays.”
• 1914: English physicists Sir W.H. Bragg and son Sir W.L. Bragg
show that the scattering of x-rays can be represented as a
"reflection” by successive planes of atoms within a crystal.
• 1915: Braggs awarded Nobel Prize
Crystals
• Crystals used in X-ray crystallography are visible to the
naked eye, they contain a vast number of precisely
ordered, identical molecules. A crystal that is 0.5 millimeters
on each side contains around 1015 medium-sized protein
molecules.

8. • When the crystals are fully formed, they are placed in a tiny
glass tube or scooped up with a loop made of nylon, glass fiber,
or other material depending on the preference of the
researcher. The tube or loop is then mounted in the X-ray
apparatus, directly in the path of the X-ray beam.
• The searing force of powerful X-ray beams can burn crystal
through a hole, if crystal left too long in their path. To minimize
radiation damage, researchers flash-freeze their crystals in
liquid nitrogen.
• Crystals for x-ray diffraction must be:
– Perfect - no twinning, inclusions or other imperfections
– small (0.1 - 0.5 mm)

9. Why do we need a crystal?
• The diffraction from a single molecule would be too weak to be
measurable. So we use an ordered three-dimensional array of
molecules, i.e. Crystal, to magnify the signal.
• Even a small protein crystal might contain a billion molecules.
• If the internal order of the crystal is poor, then the X-rays will not
be diffracted to high angles or high resolution and the data will
not yield a detailed structure.
• If the crystal is well ordered, then diffraction will be measurable
at high angles or high resolution and a detailed structure should
result.
• The X-rays are diffracted by the electrons in the structure and
consequently the result of an X-ray experiment is a 3-
dimensional map showing the distribution of electrons in the
structure.

10. X-ray crystallography
• More than 85 percent of the protein structures that are known
have been determined using X-ray crystallography.
• Crystallographers aim high-powered X-rays at a tiny crystal
containing trillions of identical molecules. The crystal scatters the
X-rays onto an electronic detector like a disco ball spraying light
across a dance floor. The electronic detector is the same type
used to capture images in a digital camera.
• After each blast of X-rays, lasting from a few seconds to several
hours, the researchers precisely rotate the crystal by entering its
desired orientation into the computer that controls the X-ray
apparatus. This enables the scientists to capture in three
dimensions how the crystal scatters, or diffracts, X-rays.

11. • The intensity of each diffracted ray is fed into a computer,
which uses a mathematical equation called a ‘Fourier
transform’ to calculate the position of every atom in the
crystallized molecule.
• The result—the researchers' masterpiece—is a three-
dimensional digital image of the molecule. This image
represents the physical and chemical properties of the
substance and can be studied in intimate, atom-by-atom detail
using sophisticated computer graphics software.

12. Steps in Structure Determination
1. Protein purification.
2. Protein crystallization.
3. Data collection.
4. Structure Solution (Phasing)
5. Structure determination (Model building and refinement)

13. Step1:Protein Purification
• Protein Purification: it is a series of processes intended to isolate one
or a few proteins from a complex mixture, usually cells, tissues or whole
organisms.
• Why Protein Purification?
– Characterization of the function.
– Structure
– Interactions of the protein.
– protein are required with better than 95% purity
Step2:Protein crystallization
Why Crystallization:
•X-ray scattering from a single unit would be weak.
•A crystal arranges a huge number of molecules in the same orientation.
•Scattered waves add up in phase and increase Signal to a level which
can be measured.
•This is often the rate-limiting step in straightforward structure
determinations, especially for membrane proteins

14. • The first-and often most difficult-step is to obtain an adequate
crystal of the material under study.
• The crystal should be sufficiently large (typically larger than 0.1
mm in all dimensions), pure in composition and regular in
structure, with no significant internal imperfections such as
cracks or twinning.
• 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.

15. X ray infrastructure

16. Characteristics of crystals
• The best crystals are pure, perfectly symmetrical, three-dimensional
repeating arrays of precisely packed molecules.
• They can be different shapes, from perfect cubes to long needles.
• Most crystals used for these studies are barely visible (less than 1
millimeter on a side).
• Proteins are difficult to crystallize because of their complexity
Methods of growing Protein crystals:
Vapor Diffusion -(Hanging Drop Method):
• This is probably the most common ways of crystallation.
• A drop of protein solution is suspended over a reservoir containing
buffer and precipitant.
• Water diffuses from the drop to the solution leaving the drop with
optimal crystal growth conditions.

17. Batch crystallization:
• A saturated protein solution left in a sealed container to let the
crystals grow.
Microbatch crystallization:
• A drop of protein solution is put in inert oil and left to grow. Here
probability is of some diffusion of proteins into the oil.
Macroseeding :
• A crystal is grown in a highly saturated solution and placed in a
less saturated one where only growth of the crystal will occur.
• Microseeding A few crystals are grown, then crushed, and put into
a final solution that combines them into a few nice crystals. This
involves quite a bit of experimentation with solutions'
concentrations to get the desired number of crystals.
Free interface diffusion: A container has levels of varying
saturation. Crystals form initially in the highly saturated part, but
as the solution mixes, it eventually only supports crystal growth.

18. Crystals MUST be:
Small in size:
•Less than 1 millimeter
PERFECT:
•No cracks
•No Inclusions, such as air bubbles
Hanging Drop Method:
•1 to 5μl protein solution is suspended
over a 1 ml reservoir containing
precipitant solution
•e.g. ammonium sulfate solution or
polyethylene glycol

19. Exposing X‐Rays:
• The crystal is placed in an intense beam of X-rays, usually of a
single wavelength (monochromatic X-rays), producing the
regular pattern of reflections. As the crystal is gradually rotated,
previous reflections disappear and new ones appear; the
intensity of every spot is recorded at every orientation of the
crystal.
• Multiple data sets may have to be collected, with each set
covering slightly more than half a full rotation of the crystal and
typically containing tens of thousands of reflections.

20. Data Collection
• The source of the X-rays is often a synchrotron.
• The typical size for a crystal for data collection may be 0.3 x 0.3 x 0.1
mm.
• The crystals are bombarded with X-rays which are scattered from the
planes of the crystal lattice.
• The scattered X-rays are captured as a diffraction pattern on a
detector such as film or an electronic device.

21. • In the third step, these data are combined computationally with
complementary chemical information to produce and refine a
model of the arrangement of atoms within the crystal.
• The final, refined model of the atomic arrangement-now called a
crystal structure is usually stored in a public database.
• After the diffraction pattern is obtained, the data is then
processed by a computer and the structure of the atom or
molecule is deduced and visualized.

22. Identification
• When X-rays are beamed at the crystal, electrons diffract the X-rays,
which causes a diffraction pattern. Using the mathematical Fourier
transform these patterns can be converted into electron density maps.
• These maps show contour lines of electron density. Since electrons
more or less surround atoms uniformly, it is possible to determine where
atoms are located.
• Unfortunately since hydrogen has only one electron, it is difficult to map
hydrogens.
• To get a three dimensional picture, the crystal is rotated while a
computerized detector produces two dimensional electron density maps
for each angle of rotation.
• The third dimension comes from comparing the rotation of the crystal
with the series of images. Computer programs use this method to come
up with three dimensional spatial coordinates.

23. • Atoms with higher atomic numbers (heavy atoms) have more
electrons and therefore diffract x-rays more effectively. Diffractive
power example: Fe > C > H.
• Hydrogen atoms often not located exactly due to small size and large
thermal motion.
• Electron density map provides location of atoms relative to each other.
– Smaller circle = higher electron density; center of circles = atom
• Bond angles, bond lengths may be determined → gives position of
atoms in space.
• Connect the dots to get the molecular structure.

25. Electron Density Map
Molecular Structure

26. USES
• Used to study many materials which form crystals like salts, metals,
minerals, semiconductors, as well as various inorganic, organic and
biological molecules.
• Determine electron density, the mean positions of the atoms in the
crystal their chemical bonds and various other information.
• Size of atoms, the lengths and types of chemical bonds, and the
atomic-scale differences among various materials, especially
minerals. The method also revealed the structure and function of
many biological molecules, including vitamins, drugs, proteins and
nucleic acids such as DNA.

27. • Characterizing the atomic structure of new materials and in
discerning materials that appear similar by other experiments.
• X-ray crystal structures can also account for unusual electronic or
elastic properties of a material, shed light on chemical interactions
and processes, or serve as the basis for designing pharmaceuticals
against diseases.
In 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.

28. 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 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.
• For 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.

29. 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.
In 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.

30. LIMITATIONS
• Must have a single, robust (stable) sample, generally between
50—250 microns in size
• Optically sample should be clear
• Twinned samples can be handled with difficulty
• Data collection generally requires between 24 and 72 hours

31. Thank You