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Crystallization Laboratory

Crystallization Laboratory. The agony and the ecstasy of protein crystallization M230D,Jan 2008. Goal: crystallize Proteinase K and its complex with PMSF. Non-specific serine protease frequently used as a tool in molecular biology. PMSF is a suicide inhibitor. Toxic!

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Crystallization Laboratory

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  1. Crystallization Laboratory The agony and the ecstasy of protein crystallization M230D,Jan 2008

  2. Goal: crystallize Proteinase K and its complex with PMSF • Non-specific serine protease frequently used as a tool in molecular biology. • PMSF is a suicide inhibitor. Toxic! • Number of amino acids: 280 • Molecular weight: 29038.0 • Theoretical pI: 8.20 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%

  3. Why are crystals so important for X-ray diffraction methods ?

  4. It is the “order” of a crystal that ultimately determines the quality of the structure. Order- describes the degree of regularity (or periodicity) in the arrangement of identical objects. One dimensional order Two dimensional order supersaturated protein solution. Three dimensional order DISORDERED ORDERED

  5. Protein crystals are ordered (periodic) arrays of protein molecules. Order- describes the degree of regularity (or periodicity) in the arrangement of identical objects. One dimensional order Two dimensional order supersaturated protein solution. Three dimensional order DISORDERED ORDERED

  6. c a b “Order” is perfect when the crystallized object is regularly positioned and oriented in a lattice.

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

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

  9. When a crystal is disordered, poor diffraction results from destructive interference of photons. Interference is destructive because path lengths differ by non integral multiple of the wavelength (nl). detector 7 6 crystal 2 9 Incident X-rays . Out of phase . Path lengths differences are not nl because of disorder.

  10. Crystal order (and resolution) improves with increasing number of lattice contacts • Trypsin (1gdn) • 0.8 Å resolution • Solvent content=36.6% • Potassium channel (1p7b) • 3.7 Å resolution • Solvent content=77.7%

  11. Lattice contacts can form only where the protein surface is rigid. By exposing rigid surface area, you enable new crystal forms previously unachievable. • Eliminate floppy, mobile termini (cleave His tags) • Express individual domains separately and crystallize separately, or… • Add a ligand that bridges the domains and locks them together. • Mutate high entropy residues (Glu, Lys) to Ala. Illustrate this.

  12. Crystallization: The task of coaxing protein molecules into a crystal.

  13. Crystallization Solubilization Is crystallization spontaneous under biological conditions? A lysozyme crystal Orientation and position of molecules are locked in a 3D array High “order” Solvated lysozyme monomers Random orientation and position

  14. The barriers to crystallization Unstable nucleus • Energy penalty • Lose 3 degrees of freedom in orientation of protein molecules • Lose 3 degrees of freedom in translation of protein molecules 1 crystal (lysozyme)N • Energy gained • Some entropy gained by freeing some surface bound water molecules. • Small enthalpic gain from crystal packing interactions. N soluble lysozyme molecules DG • Also, nucleation imposes a kinetic barrier. • Unstable because too few molecules are assembled to form all lattice contacts. nM→Mn

  15. The barriers to crystallization Unstable nucleus DG is decreased and the nucleation barrier lowered by increasing the monomer concentration [M]. 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! N soluble lysozyme molecules 1 crystal (lysozyme)N DG nM→Mn

  16. Methods for achieving 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. • We are going to dissolve lyophilized protein in a small volume of water. Concentrate protein

  17. Methods for achieving 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.

  18. Systematic vs. Shotgun Screening • Shotgun- for finding initial conditions, samples different preciptating agents, pHs, salts. • Systematic-for optimizing crystallization condtions. First commercially Available crystallization Screening kit. Hampton Crystal Screen 1

  19. Methods for achieving supersaturation. Drop =½ protein + ½ reservoir 3) Further 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!

  20. The details of the method.

  21. Practical Considerations • Begin with reservoirs: • pipet req’d amount of ammonium sulfate to each well. • Pipet req’d Tris buffers, to each well • Same with water. • Then swirl tray gently to mix. • When reservoirs are ready, lay 6 coverslips on the tray lid, • then pipet protein drops on slips and invert over reservoir. • Only 6 at a time, or else dry out. Linbro or VDX plate

  22. Proper use of the pipetor.

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

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

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

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

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

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

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

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

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

  32. 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 ||||| ||||| |||||

  33. 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

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

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

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

  37. 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!

  38. 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.

  39. 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

  40. 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

  41. Proteinase K time lapse photography • Covers first 5 hours of crystal growth in 20 minute increments 500 mm

  42. Heavy Atom Gel Shift Assay.Why?

  43. 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.

  44. Why do heavy atoms have to be screened? • To affect the diffraction pattern, heavy atom binding must be specific • Must bind the same site (e.g. Cys 134) on every protein molecule throughout the crystal. • Non specific binding does not help. • Specific binding often requires specific side chains (e.g. Cys, His, Asp, Glu) and geometry. • It is not possible to determine whether a heavy atom will bind to a protein given only its amino acid composition.

  45. Before 2000, trial & error was the primary method of heavy atom screening • Pick a heavy atom compound • hundreds to chose from • Soak a crystal • Most of the time the heavy atom will crack the crystal. • If crystal cracks, try lower concentration or soak for less time. • Surviving crystal are sent for data collection. • Collect a data set • Compare diffraction intensities between native and potential derivative. • Enormously wasteful of time and resources. Crystals are expensive to make. How many crystallization plates does it take to find a decent heavy atom derivative?

  46. Heavy Atom Gel Shift Assay • Specific binding affects mobility in native gel. • Compare mobility of protein in presence and absence of heavy atom. • Heavy atoms which produce a gel shift are good candidates for crystal soaking • Collect data on soaked crystals and compare with native. • Assay performed on soluble protein, not crystal. None Hg Au Pt Pb Sm

  47. Procedures • Just incubate protein with heavy atom for a minute. • Pipet 3 uL of protein on parafilm covered plate. • Pipet 1 uL of heavy atom as specified. • Give plate to me to load on gel. • Run on a native gel • We use PhastSystem • Reverse Polarity electrode • Room BH269 (Yeates Lab)

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