- 1 Crystallization Laboratory Why are crystals so important as long as X-ray diffraction methods Order is perfect when the crystallized object is regularly positioned in addition to oriented in a lattice. Lattice contacts can as long as m only where the protein surface is rigid. Crystallization: The task of coaxing protein molecules into a crystal.
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Crystallization Laboratory Why are crystals so important as long as X-ray diffraction methods Order is perfect when the crystallized object is regularly positioned in addition to oriented in a lattice. Lattice contacts can as long as m only where the protein surface is rigid. Crystallization: The task of coaxing protein molecules into a crystal.
Rabago, Mary, News Anchor has reference to this Academic Journal, PHwiki organized this Journal Crystallization Laboratory The agony in addition to the ecstasy of protein crystallization M230D,Jan 2008 Goal: crystallize Proteinase K in addition to 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% Why are crystals so important as long as X-ray diffraction methods
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It is the order of a crystal that ultimately determines the quality of the structure. supersaturated protein solution. One dimensional order Two dimensional order Three dimensional order Order- describes the degree of regularity (or periodicity) in the arrangement of identical objects. DISORDERED ORDERED Protein crystals are ordered (periodic) arrays of protein molecules. supersaturated protein solution. One dimensional order Two dimensional order Three dimensional order Order- describes the degree of regularity (or periodicity) in the arrangement of identical objects. DISORDERED ORDERED Order is perfect when the crystallized object is regularly positioned in addition to oriented in a lattice.
When a crystal is ordered, strong diffraction results from constructive interference of photons. Incident X-ray crystal Interference is constructive because path lengths differ by some integral multiple of the wavelength (nl). This situation is possible only because the diffracting objects are periodic. 1 2 3 4 5 1 2 3 4 5 6 detector In phase Nonregularity in orientation or position limits the order in addition to usefulness of a crystal. Rotational disorder Translational disorder Perfect order Disorder destroys the periodicity leading to Streaky, weak, fuzzy, diffraction. When a crystal is disordered, poor diffraction results from destructive interference of photons. Incident X-rays crystal Interference is destructive because path lengths differ by non integral multiple of the wavelength (nl). Path lengths differences are not nl because of disorder. 2 6 7 9 detector Out of phase .
Crystal order ( in addition to 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% Lattice contacts can as long as m only where the protein surface is rigid. Eliminate floppy, mobile termini (cleave His tags) Express individual domains separately in addition to crystallize separately, or Add a lig in addition to that bridges the domains in addition to locks them together. Mutate high entropy residues (Glu, Lys) to Ala. By exposing rigid surface area, you enable new crystal as long as ms previously unachievable. Illustrate this. Crystallization: The task of coaxing protein molecules into a crystal.
Is crystallization spontaneous under biological conditions A lysozyme crystal Orientation in addition to position of molecules are locked in a 3D array High order Solvated lysozyme monomers R in addition to om orientation in addition to position The barriers to crystallization Energy penalty Lose 3 degrees of freedom in orientation of protein molecules Lose 3 degrees of freedom in translation of protein molecules DG N soluble lysozyme molecules nMMn Energy gained Some entropy gained by freeing some surface bound water molecules. Small enthalpic gain from crystal packing interactions. Also, nucleation imposes a kinetic barrier. Unstable because too few molecules are assembled to as long as m all lattice contacts. 1 crystal (lysozyme)N Unstable nucleus The barriers to crystallization DG is decreased in addition to the nucleation barrier lowered by increasing the monomer concentration [M]. nMMn 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 in addition to into the crystal. Supersaturate! DG N soluble lysozyme molecules nMMn 1 crystal (lysozyme)N Unstable nucleus
Methods as long as achieving supersaturation. 1) Maximize concentration of purified protein Centricon-centrifugal as long as ce 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 Methods as long as 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. Systematic vs. Shotgun Screening Shotgun- as long as finding initial conditions, samples different preciptating agents, pHs, salts. Systematic- as long as optimizing crystallization condtions. First commercially Available crystallization Screening kit. Hampton Crystal Screen 1
Methods as long as achieving supersaturation. 3) Further dehydrate the protein solution Hanging drop vapor diffusion Sitting drop vapor diffusion Dialysis Liquid-liquid interface diffusion Note: Ammonium sulfate concentration is 2M in reservoir in addition to only 1M in the drop. With time, water will vaporize from the drop in addition to condense in the reservoir in order to balance the salt concentration.SUPERSATURATION is achieved! 2M ammonium sulfate Drop =½ protein + ½ reservoir The details of the method. Practical Considerations Begin with reservoirs: pipet reqd amount of ammonium sulfate to each well. Pipet reqd 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 in addition to invert over reservoir. Only 6 at a time, or else dry out. Linbro or VDX plate
Proper use of the pipetor. Which pipetor would you use as long as delivering 320 uL of liquid P1000 P200 P20 Each pipetor has a different range of accuracy P1000 P200 P20 200-1000uL 20-200uL 1-20uL
Which pipetor would you use as long as delivering 170 uL of ammonium sulfate P200 P20 How much volume will this pipetor deliver 0 2 7 How much volume will this pipetor deliver 1 7 0
How much volume will this pipetor deliver 0 2 7 What is wrong with this picture 0 2 7 – – – – 50 mL What is wrong with this picture – – – – 50 mL
Be as long as e 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 as long as less time. Surviving crystal are sent as long as data collection. Collect a data set Compare diffraction intensities between native in addition to potential derivative. Enormously wasteful of time in addition to resources. Crystals are expensive to make. How many crystallization plates does it take to find a decent heavy atom derivative Heavy Atom Gel Shift Assay Specific binding affects mobility in native gel. Compare mobility of protein in presence in addition to absence of heavy atom. Heavy atoms which produce a gel shift are good c in addition to idates as long as crystal soaking Collect data on soaked crystals in addition to compare with native. Assay per as long as med on soluble protein, not crystal. None Hg Au Pt Pb Sm Procedures Just incubate protein with heavy atom as long as 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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