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X-RAY DIFFRACTION

-SHIYAS

OUTLINE
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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 1914 1915 1953 Now X-rays discovered by Roentgen First diffraction pattern of a crystal made by Knipping and von Laue Theory to determine crystal structure from diffraction pattern developed by Bragg. DNA structure solved by Watson and Crick 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 B

A

• 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 solve the structure of DNA in 1953

Rosalind Franklin

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  Missing smears 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 structures  Missing smears indicate

Photo 51- The x-ray diffraction image that allowed Watson and Crick to solve the structure of DNA

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 indicate

Photo 51- The x-ray diffraction image that allowed Watson and Crick to solve the structure of DNA

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 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 structures  Missing smears indicate

Photo 51- The x-ray diffraction image that allowed Watson and Crick to solve the structure of DNA

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
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SELECTION OF PURE PROTEIN CRYSTALLIZATION OF PROTEIN X RAY DIFFRACTION ELECTRON DENSITY MAP REFINEMENT

SELECTION OF PURE PROTEIN

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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. substances that are not crystalline but they display some regularity of molecular structures. isotopes may be identified which determine the wave length of their characteristic line spectra.

 It can also be applied to powdered

 By this chemical elements and their

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

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