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Crystallization Laboratory

Crystallization Laboratory. The many facets of protein crystallization M230D, January 2011. Crystal structure determination pipeline. 1) chose gene product, source organism, full length, fragment, or fusion. select protein target clone express crystallize solve deposit in PDB.

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Crystallization Laboratory

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  1. Crystallization Laboratory The many facets of protein crystallization M230D, January 2011

  2. Crystal structure determination pipeline 1) chose gene product, source organism, full length, fragment, or fusion select protein target clone express crystallize solve deposit in PDB 2) chose vector, tag, location of tag (N or C?) 3) Chose host organism, temperature, media, purification scheme 4) Screen 1000 conditions Screen for crystal quality 5) collect diffraction data make heavy atom derivative determine heavy atom sites calculate map interpret map refine coordinates 6) publish

  3. Target protein sequences 84% success 99% success 8% success 49% success 93% success • Joint Center for Structural Genomics established. 2000. • Statistics reported http://www.jcsg.org/on Jan 4, 2010. Selected Targets: 33209 Cloned: 27959 Expressed: 27640 Crystallized: 2128 Solved: 1045 Deposited in PDB: 968

  4. Why is it necessary to grow crystals? Growing a suitable crystal is such a hurdle!

  5. In a crystal, the diffraction signal is amplified by the large number of repeating units (molecules). A 100 mm3 crystal contains 1012 unit cells Diffraction from a single molecule is not currently measurable. Diffraction intensity is proportional to the number of unit cells in the crystal (Darwin’s formula, 1914).

  6. c a b In a crystal, the ordered, periodic arrangement of molecules produces constructive interference.

  7. Interference is constructive because path lengths differ by some integral multiple of the wavelength (nl). detector 7 6 5 4 3 8 2 7 7 6 6 1 5 5 4 4 9 3 3 8 2 2 1 1 In phase This situation is possible only because the diffracting objects are periodic. When a crystal is ordered, strong diffraction results from constructive interference of photons. crystal Incident X-ray

  8. Irregularity in orientation or translation limits theorderand usefulness of a crystal. Translational disorder Rotational disorder Perfect order Disorder destroys the periodicity leading to Streaky, weak, fuzzy, diffraction.

  9. Irregularity in orientation or translation limits theorderand usefulness of a crystal. Translational disorder Rotational disorder Perfect order (CCML, Yeates Lab) (bacteriorhodopsin, Bowie Lab) Disorder destroys the periodicity leading to Streaky, weak, fuzzy, diffraction.

  10. What makes crystallization such a difficult challenge?

  11. Enthalpic term Entropic term DGcrystal=DHcrystal-T(DSprotein+DSsolvent) Is DHcrystal favorable? protein crystal protein in solution

  12. Yes, DHcrystal is modestly favorable (0 to -17 kcal/mol) • large area • specific • rigid protein crystal protein in solution lattice contacts

  13. Is TDSprotein favorable? protein crystal protein in solution

  14. No,TDSprotein is strongly unfavorable (+7 to +25 kcal/mol) protein crystal protein in solution • 0 degrees of freedom in orientation • 0 degrees of freedom in translation • 3 degrees of freedom in orientation • 3 degrees of freedom in translation

  15. Is TDSsolvent favorable? protein crystal protein in solution

  16. O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H Yes,TDSsolvent is favorable (-7.5 to -50 kcal/mol) protein crystal protein in solution • 3 degrees of freedom in orientation • 3 degrees of freedom in translation • 0 degrees of freedom in orientation • 0 degrees of freedom in translation

  17. DGcrystal=DHcrystal-T(DSprotein+DSsolvent) DGcrystal= -small + large – largeDGcrystal= -small

  18. or Strategies to lessen the entropic penalty, TDSprotein. • Eliminate floppy, mobile termini (cleave His tags) • Express individual domains separately and crystallize separately, or… • Add a ligand (or protein binding partners) that bridges the domains and locks them together. • Mutate high entropy residues (Glu, Lys) to Ala.

  19. Increase [protein] to favor crystallization Increasing the monomer concentration [M] pushes the equilibrium toward the product. nM→Mn DG=DGo+RTln( [Mn]/[M]n ) Lesson:To crystallize a protein, you need to increase its concentration to exceed its solubility (by 3x). Force the monomer out of solution and into the crystal. Supersaturate! Unstable nucleus N soluble lysozyme molecules 1 crystal (lysozyme)N DG nM→Mn

  20. Three steps to achieve supersaturation. 1) Maximize concentration of purified protein • Centricon-centrifugal force • Amicon-pressure • Vacuum dialysis • Dialysis against high molecular weight PEG • Ion exchange. • Slow! Avoid precipitation. Co-solvent or low salt to maintain native state. Concentrate protein

  21. Three steps to achieve supersaturation. 2)Add a precipitating agent • Polyethylene glycol • PEG 8000 • PEG 4000 • High salt concentration • (NH4)2SO4 • NaH2PO4/Na2HPO4Polyethylene glycol • Small organics • ethanol • Methylpentanediol (MPD) PEG Polymer of ethylene glycol Precipitating agents monopolize water molecules, driving proteins to neutralize their surface charges by interacting with one another. It can lead to (1) amorphous precipitate or (2) crystals.

  22. Three steps to achieve supersaturation. Drop =½ protein + ½ reservoir 3) Allow vapor diffusion to dehydrate the protein solution • Hanging drop vapor diffusion • Sitting drop vapor diffusion • Dialysis • Liquid-liquid interface diffusion 2M ammonium sulfate Note: Ammonium sulfate concentration is 2M in reservoir and only 1M in the drop. With time, water will vaporize from the drop and condense in the reservoir in order to balance the salt concentration.—SUPERSATURATION is achieved!

  23. Naomi E Chayen & Emmanuel SaridakisNature Methods - 5, 147 - 153 (2008)Published online: 30 January 2008; | doi:10.1038/nmeth.f.203 Precitating agent concentration

  24. Conventionally, try shotgun screening first, then systematic screening • Shotgun- for finding initial conditions, samples different preciptating agents, pHs, salts. • Systematic-for optimizing crystallization conditions. First commercially Available crystallization Screening kit. Hampton Crystal Screen 1

  25. The details of the experiment.

  26. Goal: crystallize Proteinase K and its complex with PMSF • Number of amino acids: 280 • Molecular weight: 29038.0 • Theoretical pI: 8.20 • Non-specific serine protease frequently used as a tool in molecular biology. • PMSF is a suicide inhibitor. Toxic! MAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASH PEFEGRAQMVKTYYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGS GQYSTIIAGMDFVASDKNNRNCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVA VAAGNNNADARNYSPASEPSVCTVGASDRYDRRSSFSNYGSVLDIFGPGTSILST WIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACRYIADTANKGDLSNIPF GTVNLLAYNNYQA Ala (A) 33 11.8% Arg (R) 12 4.3% Asn (N) 17 6.1% Asp (D) 13 4.6% Cys (C) 5 1.8% Gln (Q) 7 2.5% Glu (E) 5 1.8% Gly (G) 33 11.8% His (H) 4 1.4% Ile (I) 11 3.9% Leu (L) 14 5.0% Lys (K) 8 2.9% Met (M) 6 2.1% Phe (F) 6 2.1% Pro (P) 9 3.2% Ser (S) 37 13.2% Thr (T) 22 7.9% Trp (W) 2 0.7% Tyr (Y) 17 6.1% Val (V) 19 6.8%

  27. Reservoir Solutions • We are optimizing two types of crystals. • ProK (rows AB) • ProK+PMSF (rows CD). • There are three components to each reservoir: (NH4)2SO4, Tris buffer, and water. • We are screening six concentrations of ammonium sulfate and 2 buffer pHs. • Pipet one chemical to all reservoirs before pipeting next chemical—it saves tips. Linbro or VDX plate ( ProK ( ProK+ PMSF

  28. P20 0 2 5 ||||| Practical Considerations tray containing reservoir solutions Gently swirl tray to mix reservoir solutions. When reservoirs are ready, lay 6 coverslips on the tray lid, Then pipet protein and corresponding reservoir on slips Invert slips over reservoir. Only 6 at a time, or else dry out. tray lid tray

  29. Proper use of the pipetor.

  30. Which pipetor would you use for delivering 320 uL of liquid? P20 P200 P1000

  31. Each pipetor has a different range of accuracy P20 P200 P1000 200-1000uL 20-200uL 1-20uL

  32. P1000 Which pipetor would you use for delivering 170 uL of ammonium sulfate? P20 P200

  33. P200 How much volume will this pipetor deliver? 0 2 7 |||||

  34. P20 How much volume will this pipetor deliver? 1 7 0 |||||

  35. P1000 How much volume will this pipetor deliver? 0 2 7 |||||

  36. P1000 What is wrong with this picture? 0 2 7 ||||| - - - - 50 mL

  37. P1000 0 2 7 ||||| What is wrong with this picture? - - - - 50 mL

  38. P1000 0 2 7 ||||| Dip tip in stock solution, just under the surface. - - - - 50 mL

  39. P1000 P1000 P1000 Withdrawing and Dispensing Liquid.3 different positions Start position First stop Second stop 0 2 7 0 2 7 0 2 7 ||||| ||||| |||||

  40. P1000 Withdrawing solution: set volume, then push plunger to first stop to push air out of the tip. Start position First stop Second stop 0 2 7 ||||| - - - - 50 mL

  41. P1000 Dip tip below surface of solution. Then release plunger gently to withdraw solution Start position First stop Second stop 0 2 7 |||||

  42. P1000 To expel solution, push to second stop. Start position First stop Second stop 0 2 7 |||||

  43. P1000 When dispensing protein, just push to first stop.Bubbles mean troubles. Start position First stop Second stop 0 2 7 |||||

  44. Hanging drop vapor diffusionstep two Pipet 2.5 uL of concentrated protein (50 mg/mL) onto a siliconized glass coverslip. Pipet 2.5 uL of the reservoir solution onto the protein drop 2M ammonium sulfate 0.1M buffer BUBBLES MEAN TROUBLES Expel to 1st stop, not 2nd stop!

  45. Hanging drop vapor diffusionstep three • Invert cover slip over reservoir quickly & deliberately. • Don’t hesitate when coverslip on its side or else drop will roll off cover slip. • Don’t get fingerprints on coverslip –they obscure your view of the crystal under the microscope.

  46. Dissolving Proteinase K powder • Mix gently • Pipet up and down 5 times • Stir with pipet tip gently • Excessive mixing leads to xtal showers • No bubbles 5.25 mg ProK powder 100 uL water 4 uL of 0.1M PMSF 50 mg/mL ProK

  47. Dissolving Proteinase K powder • Mix gently • Pipet up and down 5 times • Stir with pipet tip gently • Excessive mixing leads to xtal showers • No bubbles Remove 50 uL Add to 5 uL of 100 mM PMSF 50 mg/mL ProK 55 uL of 50 mg/mL ProK+PMSF complex

  48. Proteinase K time lapse photography • illustrates crystal growth in 20 minute increments • film ends after 5 hours 500 mm

  49. Heavy Atom Gel Shift Assay.Why?

  50. Why are heavy atoms used to solve the phase problem? • Phase problem was first solved in 1960. Kendrew & Perutz soaked heavy atoms into a hemoglobin crystal, just as we are doing today. (isomorphous replacement). • Heavy atoms are useful because they are electron dense. Bottom of periodic table. • High electron density is useful because X-rays are diffracted from electrons. • When the heavy atom is bound to discrete sites in a protein crystal (a derivative), it alters the X-ray diffraction pattern slightly. • Comparing diffraction patterns from native and derivative data sets gives phase information.

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