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Dipti_X ray crystallography (1).pptx

  1. X-ray crystallography Analytical technique in plant science 32167501 Dipti yadav Roll no. – 18556051, Botany hons, Kalindi college, Delhi University Image source - 01 Your name Course, Year College logo
  2. Index 1 Introduction 2 History 3 Principle of crystallography 4 X-ray diffraction 5 Bragg’s Law 6 Application 02 Your name Course, Year College logo
  3. Introduction • X-ray crystallography is a powerful technique for visualizing the structure of protein. • It is a tool used for identifying the atomic and molecular structure of a crystal. • In crystallography the crystalline atoms cause a beam of incident X-rays to diffract into many specific directions. • Then 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. • X-ray crystallography can locate every atom in a zeolite, an aluminosilicate. 03 Botanypedia Leaningbiologymade easy
  4. History • 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. • In 1998 Dr. Peter Kim, a scientist, was able to determine the structure of a key protein responsible for the HIV infection process. 04 Botanypedia Leaningbiologymade easy
  5. X-ray 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. 05 Botanypedia Leaningbiologymade easy
  6. Principle of X-ray Crystallography • Ray diffraction by crystals is a reflection of the periodicity of crystal architecture, so that imperfection in the crystal lattice usually results in poor diffraction properties. • A crystal can be described with the aid of grid or lattice, defined by three axis and angles between them. • Along each axis a point will be repeated as distances referred to as the unit cell constants, labeled a, b and c. • Within the crystalline lattice, infinite sets of regularly spaced planes can be drawn through lattice points. • These pinlanes can be considered as the source of diffraction and are designated by a set of three numbers called the Miller indices(hkl). 06 Botanypedia Leaningbiologymade easy
  7. X-ray diffraction • X-ray crystallography uses the uniformity of light diffraction of crytals to determine the structure of molecule or atom • Then X-ray beam is used to hit the crystallized molecule. • The electron surrounding the molecule diffract as the X-rays hit them. • This forms a pattern. This type of pattern is known as X-ray diffraction pattern 07 Botanypedia Leaningbiologymade easy
  8. Bragg’s Law Bragg’s Law was introduced by Sir W.H. Bragg and his son Sir W.L. Bragg. The law states that when the x-ray is incident onto a crystal surface, its angle of incidence θ, will reflect back with a same angle of scattering θ. And, when the path difference, dd is equal to a whole number, nn, of wavelength, a constructive interference will occur. Consider a single crystal with aligned plane of lattice points separated by a distance d. Monochromatic X-rays A, B, and C are incident upon the crystal at an angle θ. They reflect off atoms X, Y, or Z. 08 Botanypedia Leaningbiologymade easy
  9. Bragg’s Law nλ = 2d sinƟ 09 Botanypedia Leaningbiologymade easy
  10. Bragg’s Law • Crystals are regular arrays of atoms, and X-rays can be considered waves of electromagnetic radiation. Atoms scatter X-ray waves, primarily through the atoms' electrons. • X-ray striking an electron produces secondary spherical waves emanating from the electron. This phenomenon is known as elastic scattering, and the electron is known as the scatterer. A regular array of scatterers produces a regular array of spherical waves. • Although these waves cancel one another out in most directions through destructive interference, they add constructively in a few specific directions, determined by Bragg's law • X-rays are used to produce the diffraction pattern because their wavelength λ is typically the same order of magnitude (1–100 angstroms) as the spacing d between planes in the crystal. In principle, any wave impinging on a regular array of scatterers produces diffraction, as predicted first by Francesco Maria Grimaldi in 1665. 10 Botanypedia Leaningbiologymade easy
  11. Thomson scattering • The X-ray scattering is determined by the density of electrons within the crystal. • Since the energy of an X-ray is much greater than that of a valence electron, the scattering may be modeled as Thomson scattering, the interaction of an electromagnetic ray with a free electron • The intensity of Thomson scattering for one particle with mass mand charge q is: 11 Botanypedia Leaningbiologymade easy
  12. • Generally a typical x-ray diffraction contain below parts: 1.Detector 2.X-ray source 3.Crystal on the end of mounting needle 4.Liquid nitrogen steam to keep crystal cold 5.Movable mount to rotate crystal 12 Botanypedia Leaningbiologymade easy
  13. T ypesofX-raydevice used Elecrons are responsible for the diffraction and intensity in crystallography Electrons they scatter x-rays weaker than heavy elements. Knowing this, protein crystallographers use high intensity x-ray sources suchas a rotating anode tube or a strong synchrotron x-ray source for analyzing the protein crystals. 13 Botanypedia Leaningbiologymade easy
  14. First step The process begins by crystallizing a protein of interest. 4 critical stepsare taken to achieve protein crystallization: Purify the protein. Determine the purity of the protein and if not pure(usually >99%), then must undergo further purification. Protein must be precipitated by dissolving it in an appropriate solvent(water- buffer soln. w/ organic salt such as 2-methyl-2,4-pentanediol). The solution has to be brought to supersaturation by adding a salt to the concentrated solution of the protein. Let the actual crystals grow. Since nuclei crystals are formed this will lead to obtaining actual crystal growth. 14 Botanypedia Leaningbiologymade easy
  15. Second Step X-rays are generated and directed toward the crystallized protein Then, the x-rays are shot at the protein crystal resulting in some of thex-rays going through the crystal and the rest being scattered in variousdirections. The crystal is rotated so that the x-rays are able to hit the protein fromall sides and angles. The pattern on the emulsion due to scattering reveals much information about the structure of the protein. The intensities of the spots and their positions are thus are thebasic experimental data of the analysis. 15 Botanypedia Leaningbiologymade easy
  16. Third Step An electron density map is created based on the measured intensities of the diffraction pattern on the film A Fourier Transform can be applied to the intensities on the filmto reconstruct the electron density distribution of the crystal The mapping gives a three-dimensional representation of the electron densities observed through the x-ray crystallography When interpreting the electron density map, resolution needs to be taken into account A resolution of 5Å - 10Å can reveal the structure of polypeptide chains, 3Å- 4Å of groups of atoms, and 1Å - 1.5Å of individual atoms. 16 Botanypedia Leaningbiologymade easy
  17. Applications of X-ray Crystallography HIV- Scientists also determined the X-ray crystallographic structure of HIV protease, a viral enzyme critical in HIV’s life cycle, in 1989. Pharmaceutical scientists hoped that by blocking this enzyme, they could prevent the virus from spreading in the body. By feeding the structural information into a computer modeling program, they could use the model structure as a reference to determine the types of molecules that might block the enzyme. Arthritis- To create an effective painkiller in case of arthritis that doesn’t cause ulcers, scientists realized they needed to develop new medicines that shut down COX-2 but not COX-1. Through structural biology, they could see exactly why Celebrex plugs up COX-2 but not COX-1 17 Botanypedia Leaningbiologymade easy
  18. Applications of X-ray Crystallography Applications of X-Ray Crystallography in Dairy Science X-ray crystallography technique has been a widely used tool for elucidation of compounds present in milk and other types of information obtained through structure function relationship. Stewart has shown that even solutions tend to assume an orderly arrangement of groups within the solution. Hence, liquid milk should, and does show some type of arrangement. The mineral constituent and lactose are the only true crystalline constituents in dairy products that can be analyzed by X-ray. Analysis of Milk Stones X-ray diffraction technique has also been applied for analysing the chemical composition of milk stones. Since each chemical compound gives a definite pattern on a photographic film according to atomic arrangement, X-rays can be used for qualitative chemical analysis as well as structural analysis. 18 Botanypedia Leaningbiologymade easy
  19. Applications of X-ray Crystallography X-Ray Analysis of MilkPowder This technique has also been used in study of milk powder. Most work hasbeen confined to determine the effect of different milk powdering processes upon structural group spacings within the milk proteins. Differentiation of Sugar Since each crystalline compound gives a definite pattern according to the atomic arrangement, the identification and the differentiation of the common sugars (sucrose, dextrose and lactose) is made simple by X-rays In case of new material X-ray crystallography is still the chief method for characterizing the atomic structure of new materials and in discerning materials that appear similar by other experiments. 19 Botanypedia Leaningbiologymade easy
  20. References: • Hickman, A. B. and Davies, D. R. (2001). Principles of macromolecular X-ray crystallography. • Current Protocols in Protein Science, Chapter 17, Unit 17.3. New York: Wiley Interscience. • Miao, J., Ishikawa, T., Shen, Q. and Earnest, T. (2008). Extending X-ray crystallography to allow the imaging of noncrystalline materials, cells, and single protein complexes. Annual Reviews in Physical Chemistry, 59, 387–410. • Mueller, M., Jenni, S. and Ban, N. (2007). Strategies for crystallization and structure determination of very large macromolecular assemblies. Current Opinion in Structural Biology, 17, 572–579. • Wlodawer, A., Minor, W., Dauter, Z. and Jaskolski, M. (2008). Protein crystallography for noncrystallographers, or how to get the best (but not more) from published macromolecular structures. FEBS Journal, 275, 1–21. • WEBSITES 20 Botanypedia Leaningbiologymade easy
  21. Thank you 21 Botanypedia Leaningbiologymade easy