X-ray crystallography is a powerful technique used in determining the three-dimensional structure of molecules at atomic resolution. It involves the use of X-rays to probe the arrangement of atoms in a crystal lattice. The information obtained from X-ray crystallography can be used to understand the function of biomolecules such as proteins, DNA, and RNA.

1. APPLICATION OF X RAY
CRYSTALLOGRAPHY IN PROTEIN
PRIDICTION
DEPARTMENT OF PHARMACOLOGY
CENTRAL UNIVERSITY OF PUNJAB, BATHINDA
SUBMITTED BY:
RIYA RAJESH GAGNANI

2. INTRODUCTION TO X-RAY
CRYSTALLOGRAPHY
• X-ray crystallography is a powerful technique used in
determining the three-dimensional structure of molecules at
atomic resolution. It involves the use of X-rays to probe the
arrangement of atoms in a crystal lattice. The information
obtained from X-ray crystallography can be used to
understand the function of biomolecules such as proteins,
DNA, and RNA.

4. HISTORY
• Wilhelm Rontgen discovered X-rays in 1895.
• The first successful application of X-ray crystallography in protein structure determination was
reported in 1958 by John Kendrew and his colleagues. Since then, X-ray crystallography has become
the most widely used method for determining the structures of proteins
• 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.

5. NOBLE PRIZE RELATED TO X-RAY CRYSTALLOGRAPHY
Year Laureate Prize Rationale
1914 Max von Laue Physics
"For his discovery of the diffraction of
X-rays by crystals, an important step in
the development of X-ray
spectroscopy.
1915
William Henry Bragg
Physics
"For their services in the analysis of
crystal structure by means of X-rays"
William Lawrence Bragg
1962
Max F. Perutz
Chemistry
"for their studies of the structures of
globular proteins"
John C. Kendrew
1962
James Dewey Watson
Medicine
"For their discoveries concerning the
molecular structure of nucleic acids
and its significance for information
transfer in living material"
Francis Harry Compton Crick
Maurice Hugh Frederick Wilkins
2003 Roderick MacKinnon Chemistry
"For discoveries concerning channels
in cell membranes for structural and
mechanistic studies of ion channels"
2009 Venkatraman Ramakrishnan Chemistry
"For studies of the structure and
function of the ribosome"
2012 Brian Kobilka Chemistry
"For studies of structure of G-protein-
coupled receptors"

6. PRINCIPLE
• The aim of x ray crystallography is to obtain a three
dimensional molecular structure from a crystal.
• A purified sample at high concentration is crystallised and
the crystals are exposed to an x ray beam.
• The resulting diffraction patterns can then be processed,
initially to yield information about the crystal packing
symmetry and the size of the repeating unit that forms the
crystal.

7. BRAGG'S LAW
 There is a definite relationship between the angle at which a
beam of X-ray fall on the parallel planes of atoms in a
crystal in order that there be strong reflection, the
wavelength of the X-rays, and the distance between the
crystal planes.
 According to Braggs law, Diffraction occurs only when the
path length of the rays reflected from successive planes
differ by a complete no of wavelengths
• Here, d is the spacing between diffracting planes,
• ɵ is the incident angle,
• n is the integer, and
• ⅄ is the wavelength of the beam
Incident X ray strikes a set of planes (with
an interplanar distance d at an angle θ.
n⅄ = 2dsinɵ

8. INSTRUMENTATION
Generally, a typical x-ray diffraction contain
below parts :
• X-ray source
• Detector
• Crystal on the end of mounting needle
• Liquid nitrogen steam to keep crystal cold
• Movable mount to rotate crystal
• Collimator system

9. APPLICATIONS OF X RAY CRYSTALLOGRAPHY

10. PROTEIN STRUCTURE
DETERMINATION
• Protein structure determination refers to
finding the exact orientations and
arrangements of different amino acids
present in the protein.
• X ray crystallography helps us to
determine the structure of proteins
which further helps us to even
determine its functions.

11. PROTEIN INTERACTION
STUDIES
• Protein interaction refers to the way in
which two or more proteins interact with
each other.
• Studies includes the orientation, site of
action and the major amino acids (of
protein) taking part in a particular
reaction and this technique helps to
determine there interactions.

12. CONFORMATIONAL STUDIES
• Conformational studies refers to the special
arrangements of atom in a molecule that
can come through free rotation of atoms in
a chemical bond.
• It is necessary to determine the
arrangements as it determine the structure
and function of proteins and this technique
is suitable to determine it.

13. ENZYME CATALYSIS
DETERMINATION
Determination of structure and type
of amino acids present in active
sites determines the catalytic
activities and interaction level of
enzymes.
This technique helps us to
determine and predict the catalytic
efficiency of enzymes.

14. X-RAY CRYSTALLOGRAPHY OF PROTEINS
Proteins are among the many biological molecules that are used for x-ray
Crystallography studies. They are involved in many pathways in biology, often catalyzing
reactions by increasing the reaction rate. Most scientists use x-ray Crystallography to
solve the structures of protein and to determine functions of residues, interactions with
substrates, and interactions with other proteins or nucleic acids. Proteins can be co -
crystallized with these substrates, or they may be soaked into the crystal after
crystallization.

15. This is the structure of APS Kinase co- crystallized with
ligands ADP and APS created via pymol by an undergrad
working in the Structural Biology lab at UC Davis;
This is the mutant overlay of APS kinase. The teal is the wild -
type and the lime green is the mutant. D63 (from the wild-type)
is mutated to asparagine.

16. PROTEIN CRYSTALLIZATION
Proteins will solidify into crystals under certain
conditions. These conditions are usually made up of
salts, buffers, and precipitating agents. This is often
the hardest step in x-ray crystallography. Hundreds of
conditions varying the salts, pH, buffer, and
precipitating agents are combined with the protein in
order to crystallize the protein under the right
conditions. This is done using 96 well plates; each
well containing a different condition and crystals;
which form over the course of days, weeks, or even
months.
Crystals of APS Kinase D63N from Penicillium chrysogenum