Week 2 - review of chemistry, intro to crystallography


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  • Structural analysis overview
  • Week 2 - review of chemistry, intro to crystallography

    1. 1. The Structures of Atoms Found in Organisms
    2. 2. Different Types of Covalent Bonds the shared electrons glue the atoms together
    3. 4. Basic Structure of An Amino Acid
    4. 5. alanine arginine asparagine aspartate cysteine glutamate glutamine glycine histidine isoleucine leucine lysine methionine phenylalanine histidine serine threonine tryptophan tyrosine valine The Twenty Amino Acids
    5. 6. Proteins are Polymers of Amino Acids some proteins in your body have the same amino acid sequence as those found in the chimpanzee
    6. 8. Critical Stages in an X-ray Analysis <ul><li>Cloning, over-expression, and protein purification </li></ul><ul><li>Crystallization </li></ul><ul><li>Data collection and processing </li></ul><ul><li>Interpretation of electron density maps </li></ul>
    7. 9. Fundamentals of Protein Crystallization <ul><li>Crystallization is simply achieved by reducing the solubility of the protein under conditions where the stable state becomes the crystalline lattice. </li></ul><ul><li>The problem is finding those conditions! </li></ul><ul><li>Must determine the appropriate precipitant and state of the protein for crystallization to occur. </li></ul>
    8. 10. Finding Crystallization Conditions <ul><li>This is still a matter of trial and error. </li></ul><ul><li>There is no guarantee that a given protein can be crystallized. </li></ul><ul><li>Crystallization screens have been developed that improve the chances of finding suitable conditions. </li></ul><ul><li>A large number of crystallization screens are commercially available from Hampton Research. </li></ul>
    9. 11. Important Parameters in Protein Crystallization <ul><li>The state and purity of the protein </li></ul><ul><li>Precipitant (type and concentration) </li></ul><ul><li>pH - usually test in 1/2 pH units </li></ul><ul><li>Temperature </li></ul><ul><li>Protein concentration </li></ul>
    10. 12. Common Precipitants (Salts) <ul><li>Salts </li></ul><ul><ul><li>ammonium sulfate </li></ul></ul><ul><ul><li>ammonium phosphate </li></ul></ul><ul><ul><li>sodium/potassium phosphate </li></ul></ul><ul><ul><li>sodium citrate </li></ul></ul><ul><ul><li>sodium sulfate </li></ul></ul><ul><ul><li>sodium chloride </li></ul></ul><ul><ul><li>magnesium sulfate </li></ul></ul>
    11. 13. Common Precipitants (Organics ) <ul><li>polymeric precipitants </li></ul><ul><ul><li>poly(ethylene glycol) 6000-8000 </li></ul></ul><ul><ul><li>methylether poly(ethylene glycol) 5000 </li></ul></ul><ul><ul><li>poly(ethylene glycol) 1000-2000 </li></ul></ul><ul><ul><li>poly(ethylene glycol) 400 </li></ul></ul><ul><li>alcohols </li></ul><ul><ul><li>ethanol, propanol </li></ul></ul><ul><ul><li>2-methyl-2,4,-pentane diol (MPD), propanediol </li></ul></ul>
    12. 14. Vapor Diffusion <ul><li>Introduced in the late 1960s </li></ul><ul><li>“ Conventional” sitting drop method was introduced by Bob Bock in 1968 (former Dean of the Graduate School at UW) </li></ul><ul><li>Hanging drop method was developed in 1971 </li></ul><ul><li>This became the standard method for crystallization by 1972 </li></ul>
    13. 15. Hanging Drop Method of Vapor Diffusion This is the most commonly used technique for surveying and growing crystals for structural analyses.
    14. 16. X-ray Diffraction is the Best Test for Crystallinity <ul><li>Most protein crystals are grown with the goal of determining their structure, thus an x-ray diffraction experiment is considered to be the ultimate proof that a well ordered crystalline lattice has been achieved. </li></ul>
    15. 17. Common Table Salt
    16. 18. Common Table Sugar
    17. 19. C-3 ’ -Methyltransferase <ul><li>Involved in the production of an unusual sugar called tetronitrose </li></ul><ul><li>Tetronitrose is found in an antitumor and antibacterial agent called tetrocarcin A. </li></ul>
    18. 21. C-3 ’ -Methyltransferase
    19. 22. S -Adenosylmethionine <ul><li>Discovered in Italy in 1952 </li></ul><ul><li>Referred to as SAMe in the vitamin supplement stores </li></ul><ul><li>May be helpful in the treatment of depression, osteoarthritis, fibromyalgia, liver disease, Alzheimer’s Disease </li></ul>
    20. 23. Space-Filling
    21. 24. Ball-and-Stick
    22. 25. Ribbon Representation
    23. 26. Electron Density
    24. 27. His 181 Glu 224 Tyr 222