X-RAY DIFFRACTION

-SHIYAS
 OUTLINE

 Introduction
 History
 How Diffraction Works
 Solving DNA
 X RAY Crystallography
 Applications
 Summary and Conclusions
 INTRODUCTION

• X-ray diffraction is used to obtain structural

information about crystalline solids.
• Useful in biochemistry to solve the 3D
structures of complex biomolecules.

 X-ray diffraction is important for:

• Solid-state physics
• Biophysics
• Chemistry and Biochemistry

X RAY
DIFFRACTOMETER
History of X-Ray Diffraction

1895 X-rays discovered by

Roentgen
1914 First diffraction
pattern of a crystal
made by Knipping and
von Laue
1915 Theory to determine
crystal structure from
diffraction pattern
developed by Bragg.
1953 DNA structure solved
by Watson and Crick
Now Diffraction improved
by computer
technology; methods
used to determine
atomic structures and
THE FIRST X-RAY
Experimental Setup
How Diffraction Works
 How Diffraction Works

 Wave Interacting with a Single Particle

 Incident beams scattered uniformly in all
directions
 Wave Interacting with a Solid
 Scattered beams interfere constructively in some
directions, producing diffracted beams
 Random arrangements cause beams to randomly
interfere and no distinctive pattern is produced
 Crystalline Material
 Regular pattern of crystalline atoms produces
regular diffraction pattern.
 Diffraction pattern gives information on crystal
structure
 Bragg’s Law

C
A

B
• Similar principle to multiple slit
experiments
• Constructive and destructive
interference patterns depend on
lattice spacing (d) and wavelength
of radiation (λ)
• By varying wavelength and
observing diffraction patterns,
information about lattice spacing is
obtained
How Diffraction Works
 Solving the Structure of DNA: History

 Rosalind Franklin- physical

chemist and x-ray
crystallographer who first
crystallized and
photographed B­DNA
 Watson & Crick- chemists
who combined the
information from Photo 51
with molecular modeling to Rosalind
solve the structure of DNA Franklin
in 1953
Solving the Structure of DNA

 Photo 51 Analysis
 “X” pattern
characteristic of
helix
 Diamond shapes
indicate long,
extended molecules
 Smear spacing
reveals distance
between repeating
structures
 Missing smears Photo 51- The x-ray diffraction
image that allowed Watson and
indicate interferenceCrick to solve the structure of DNA
from second helix
Solving the Structure of DNA

 Photo 51 Analysis
 “X” pattern
characteristic of
helix
 Diamond shapes
indicate long,
extended
molecules
 Smear spacing
reveals distance
between
repeating Photo 51- The x-ray diffraction
structures image that allowed Watson and
 Missing smears Crick to solve the structure of DNA
indicate
Solving the Structure of DNA

 Photo 51 Analysis
 “X” pattern
characteristic of
helix
 Diamond shapes
indicate long,
extended
molecules
 Smear spacing
reveals distance
between repeating Photo 51- The x-ray diffraction
structures image that allowed Watson and
Crick to solve the structure of DNA
 Missing smears
indicate
Solving the Structure of DNA

 Photo 51 Analysis
 “X” pattern
characteristic of
helix
 Diamond shapes
indicate long,
extended molecules
 Smear spacing
reveals distance
between repeating
structures Photo 51- The x-ray diffraction
image that allowed Watson and
 Missing smears
Crick to solve the structure of DNA
indicate interference
from second helix
Solving the Structure of DNA

 Photo 51 Analysis
 “X” pattern
characteristic of
helix
 Diamond shapes
indicate long,
extended
molecules
 Smear spacing
reveals distance
between repeating Photo 51- The x-ray diffraction
structures image that allowed Watson and
Crick to solve the structure of DNA
 Missing smears
indicate
Solving the Structure of DNA
 Information Gained from
Photo 51
 Double Helix
 Radius: 10 angstroms
 Distance between
bases: 3.4 angstroms
 Distance per turn: 34
angstroms
 Combining Data with
Other Information
 DNA made from:
sugar
phosphates
4 nucleotides
(A,C,G,T)
 Chargaff’s Rules
 %A=%T
 %G=%C
 Molecular Modeling
Watson and Crick’s
 X-RAY CRYSTALLOGRAPHY

 SELECTION OF PURE PROTEIN

 CRYSTALLIZATION OF PROTEIN
 X RAY DIFFRACTION
 ELECTRON DENSITY MAP
 REFINEMENT
SELECTION OF PURE
PROTEIN
 Some protein crystals grown by a variety of techniques and
using a
 number of different precipitating agents. They are
 (a) deer catalase,
 (b) trigonal form offructose-1,6-diphosphatase from chicken
liver,
 (c) cortisol binding protein from guinea pig sera,
 (d) concanavalin B from jack beans,
 (e) beef liver catalase,
 (f ) an unknown protein from pineapples,
 (g) orthorhombic form of the elongation factor Tu from
Escherichia coli,
 (h) hexagonal and cubic crystals of yeast phenylalanine
tRNA,
 (i), monoclinic laths of the gene 5 DNA unwinding protein
from bacteriophage fd,
 ( j) chicken muscle glycerol-3-phosphate dehydrogenase,
CRYSTALLIZATION OF
PROTEIN
X RAY DIFFRACTION
 ELECTRON DENSITY MAP

 Intensity and phase information

 3D image of molecule
 Uses computers graphic sytem
 3D image of electron density and
computer generated atomic model
are superimposed
 REFINEMENT

 FINAL STEP
 USES MATHEMATICAL METHODS
 REPEATED 100 OF TIMES.
 USES OF X RAY
DIFFRACTION
 Using x ray diffraction method
,crystalline substances and their
structures determined.

 It can also be applied to powdered

substances that are not crystalline
but they display some regularity of
molecular structures.

 By this chemical elements and their

isotopes may be identified which
determine the wave length of their
characteristic line spectra.
 USES OF X RAY
DIFFRACTION
 Biological structure are crystalline in
nature.x ray crystallography is the
only method to study these structures.
 Examples:
 X-ray crystal structures of proteins,
nucleic acids and other biological
molecules have been determined.

 X-ray crystallography is now used

routinely by scientists to determine
how a pharmaceutical interacts with
its protein target and what changes
might be advisable to improve it
 Franklins x-ray diffraction studies
shows that the DNA could exist in two
separate forms.
 Application in metallurgy and
mineralogy.
metallurgy e.g.:- An alloy Mg2Sn lead
to governing the structure and
stability of complex ionic crystals.
mineralogy e.g.:- X-ray
crystallographic study of the silicates.
DIFFRACTION PATTERN OF
CRYSTALLISED ENZYME
 Summary and Conclusions
 X-ray diffraction is a technique for analyzing
structures of biological molecules
 X-ray beam hits a crystal, scattering the
beam in a manner characterized by the
atomic structure
 Even complex structures can be analyzed by
x-ray diffraction, such as DNA and proteins
 This will provide useful in the future for
combining knowledge from physics,
chemistry, and biology
THANK YOU