X-RAY

DIFFRACTIO
N

PREPARED BY: AR JUN REY G.

GENILZA
INTRODUCTION
WHAT IS X-RAY DIFFRACTION???
WHAT IS DIFFRACTION???
X-RAY DIFFRACTION

In 1912 Knipping and Frinedricb in Laue's laboratory performed the first X-ray
diffraction experiment with a crystal. Their result showed at the same time that X-rays
behave as waves and that crystals display a high degree of order in arranging their
atoms, ions or molecules. The crystal acts as a three-dimensional grating and diffracts X
rays just as visible light is diffracted by an optical grating. Since 1912, X-ray
Crystallography has had an enonimous impact on chemistry and biology.
FIBER DIFFRACTION

 X-ray fiber diffraction is a technique that enables structure determination of fibrous

or polymeric molecules based on the ordered arrangement of molecular structure
along a fiber axis. Diffraction patterns are generated by the X-ray scattering from the
molecular organization of the fibrous molecule. The fibrous structures are generally
cylindrically symmetric around an uniaxial direction, which results in diffraction data
with a characteristic four quadrant symmetry, with diffraction signals arranged
symmetrically across the “meridian” (usually vertical) and “equator” (horizontal).
FIBER DIFFRACTION
CRYSTAL DIFFRACTION (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.
CRYSTAL DIFFRACTION (CRYSTALLOGRAPHY)

 X-ray crystallography is related to several other methods for determining atomic structures. Similar
diffraction patterns can be produced by scattering electrons or neutrons, which are likewise
interpreted by Fourier transformation. If single crystals of sufficient size cannot be obtained, various
other X-ray methods can be applied to obtain less detailed information; such methods include fiber
diffraction, powder diffraction and (if the sample is not crystallized) small-angle X-ray
scattering (SAXS). If the material under investigation is only available in the form
of nanocrystalline powders or suffers from poor crystallinity, the methods of electron
crystallography can be applied for determining the atomic structure.
CRYSTAL DIFFRACTION (CRYSTALLOGRAPHY)
The important information in crystal diffraction studies is just how the atoms are arranged within each unit cell,
for that arrangement describes It is now appropriate to review the steps that must be taken to determine the
three-dimensional structure of a macromolecule from crystal diffraction studies:

I. Obtain satisfactory crystals. This step is often the hardest part of the procedure, for the crystals must be of
good quality and at least a few tenths of a millimeter in minimum dimension. Crystals that are too small
will not give sharp diffraction patterns. Getting macro-molecules to crystallize well is still more of an art
than a science.
II. Record the diffraction pattern from the crystal, and measure the intensities of many of the spots.
III. Find some way to make isomorphous replacements in the molecule. Usually two or more replacements
are required.
IV. Repeat steps 1 and 2 for each isomorphous derivative.
V. Calculate structure factors and, from them, the electron density distribution. These calculations are usually
done on a large computer.
INSTRUMENT
 The beams diffracted by crystals of small compounds
are usually detected and measured with a
diffractometer. Using this instrument, the crystal can be
rotated around three or four axes and can reach nearly
every orientation. The diffracted beams are measured
with a photon counter. Because the beams are
measured one after the other, this process is slow and
not very suitable for protein crystals where thousands of
beams must be measured. Protein crystallographers
prefer an instrument that has one or more axes to
orient the crystal and is equipped with an area detector
on which hundreds or thousands of diffracted beams
are registered nearly simultaneously. A popular type of
the imaging plate is covered with phosphorescent
material. After registration, the information collected on
the imaging plate is replaced by a laser and read with a
photomultiplier.
APPLICATION

Protein crystallography is the youngest branch of X-ray crystallography. It started in 1934 when Bernal
took the first X-ray diffraction picture of a protein crystal, first a crystal of pepsin, soon followed by a
crystal of insulin. These observations showed that even these large molecules are nicely ordered in their
crystals and that their structures might be solved. This was not an easy problem, however, and it took
twenty years to find a solution. Surprisingly, it could be done by a technique already successfully applied
for small organic compounds the method of isomorphous replacement in which a heavy atom is attached
to the protein structure. It was Perutz' achievement to show that the method could also be used for
proteins and that the heavy atoms cause a much larger change in intensity than initially expected.
Because of the rather primitive instrumentation, progress was slow at first. With the introduction of
more sophisticated instrumentation and structures are being published with ever increasing speed. They
are collected in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.