X Ray Seminar

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 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

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 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     

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

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