Crystallization Laboratory Putting crystals in perspective. Why do I need them
Rabago, Mary, News Anchor has reference to this Academic Journal, PHwiki organized this Journal Crystallization Laboratory Putting crystals in perspective. Why do I need them How do I grow them M230D, January 2012 Crystallization is one step in the structure determination pipeline select protein target clone express crystallize solve deposit in PDB 1) chose gene product, source organism, full length, fragment, or fusion 2) chose vector, tag, location of tag (N or C) 3) Chose host organism, temperature, media, purification scheme 4) Screen 1000 conditions Screen as long as crystal quality 5) collect diffraction data make heavy atom derivative determine heavy atom sites calculate map interpret map refine coordinates 6) publish Joint Center as long as Structural Genomics established. 2000. Statistics reported http://www.jcsg.org/ on Jan 4, 2010. Deposited in PDB: 968 Crystallized: 2128 Solved: 1045 Expressed: 27640 Cloned: 27959 Selected Targets: 33209
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Why is it necessary to grow crystals Growing a suitable crystal is such a hurdle! In a crystal, the diffraction signal is amplified by the large number of repeating units (molecules). Diffraction intensity is proportional to the number of unit cells in the crystal (Darwins as long as mula, 1914). Diffraction from a single molecule is not currently measurable. A 100 mm3 crystal contains 1012 unit cells In a crystal, the ordered, periodic arrangement of molecules produces constructive interference (i.e. amplification of diffraction signal).
When a crystal is ordered, strong diffraction results from constructive interference of photons. Incident X-ray crystal Irregularity in orientation or translation 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. Irregularity in orientation or translation 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. (CCML, Yeates Lab) (bacteriorhodopsin, Bowie Lab)
What makes crystallization such a difficult challenge DGcrystal=DHcrystal-T(DSprotein+DSsolvent) protein crystal protein in solution Is DHcrystal favorable Enthalpic term Entropic term Yes, DHcrystal is modestly favorable (0 to -17 kcal/mol) large area specific rigid lattice contacts protein crystal protein in solution
Is TDSprotein favorable protein crystal protein in solution No, TDSprotein is strongly unfavorable (+7 to +25 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 Is TDSsolvent favorable protein crystal protein in solution
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 DGcrystal=DHcrystal-T(DSprotein+DSsolvent) DGcrystal= -small + large large DGcrystal= -small Strategies to lessen the entropic penalty, TDSprotein. Eliminate floppy, mobile termini (cleave His tags) Express individual domains separately in addition to crystallize separately, or Add a lig in addition to (or protein binding partners) that bridges the domains in addition to locks them together. Mutate high entropy residues (Glu, Lys) to Ala. (http://services.mbi.ucla.edu/SER/)
Increase [protein] to favor crystallization DG N soluble lysozyme molecules nMMn 1 crystal (lysozyme)N Increasing the monomer concentration [M] pushes the equilibrium toward the product. 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! Unstable nucleus Three steps to achieve 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. Concentrate protein 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.
Three steps to achieve supersaturation. 3) Allow vapor diffusion to 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 Naomi E Chayen & Emmanuel Saridakis Nature Methods – 5, 147 – 153 (2008)Published online: 30 January 2008; doi:10.1038/nmeth.f.203 Precipitating agent concentration Conventionally, try shotgun screening first, then systematic screening Shotgun- as long as finding initial conditions, samples different preciptating agents, pHs, salts. Systematic- as long as optimizing crystallization conditions. First commercially Available crystallization Screening kit. Hampton Crystal Screen 1
Heavy Atom Gel Shift Assay. Why 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 in addition to derivative data sets gives phase in as long as mation. 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) in addition to geometry. It is not possible to determine whether a heavy atom will bind to a protein given only its amino acid composition.
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 is 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 (100 mM) 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)
Preparing Proteinase K -PMSF complex Mix gently Pipet up in addition to down <5 times Stir with pipet tip gently Excessive mixing leads to xtal showers No bubbles 36 mL of 50 mg/mL ProK Remove 18 uL of proteinase K in addition to add to PMSF 5 mL of 100 mM PMSF Proteinase K time lapse photography illustrates crystal growth in 20 minute increments film ends after 5 hours 500 mm
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