Methods for Structure Determination

|
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
 8 views
of 47

Please download to get full document.

View again

Description
Methods for Structure Determination. Chemistry and Chemical Biology Rutgers University. How are macromolecular structures determined?. X-ray (X-ray crystallography). NMR (Nuclear Magnetic Resonance). EM (Electron Microscopy). Protein Data Bank. Download. X-ray Crystallography.
Share
Transcript
Methods for Structure DeterminationChemistry and Chemical BiologyRutgers UniversityHow are macromolecular structures determined?X-ray(X-ray crystallography)NMR(Nuclear Magnetic Resonance)EM(Electron Microscopy)Protein Data BankDownloadX-ray CrystallographyTarget selectionProtein productionCrystallizationStructure calculationRefinementValidationDeposition to PDBAnnotationData collectionPhasinghttp://www-structmed.cimr.cam.ac.uk/Course/Overview/Overview.html#methodsSome Common Concepts
  • Symmetry: Translation, Rotation, Reflection, Inversion
  • Crystals: Lattice, Unit cell, Asymmetric Unit
  • Diffraction: Light diffraction, X-ray diffraction, diffraction patterns
  • Real vs Reciprocal space: Fourier transform
  • Phase problem: What is it? Phasing strategies
  • Structure solution pipeline
  • SymmetryTranslationRotation (pt)Inversion?? (pt)Mirror (pt)M.C. EscherMineralProteinCrystals,,,,Unit Cell 1,,,,,,,,,,,,,,,,,,,,,,,,Convolution,,,,,,,,,,,,Crystal structure,,,,,,,,Unit Cell 2Lattice, Crystal and Unit celllattice................,objectMacromolecular Crystal LatticeAlexander McPherson, Introduction to Macromolecular Crystallography Wiley-Liss, 2002Unit Cell and Asymmetric UnitSymmetry in Crystals1
  • 1-fold
  • 2-fold
  • 3-fold
  • 4-fold
  • 6-fold
  • 2345-, 7-, 8- and higher fold symmetries6do not pack in a crystalCrystal SystemsZ. Dautera & M. Jaskolskibhttp://journals.iucr.org/j/issues/2010/05/02/kk5061/kk5061bdy.htmlThe International TablesDiffractionSunrise through a screened windowhttp://www.flickr.com/photos/fizzix/2458009067/in/photostream/Diffraction in Actionhttp://mrsec.wisc.edu/Edetc/supplies/DNA_OTK/images/ABCH.movVsDirect/Real space Reciprocal spaceDiffraction patternReciprocal lattice (a*, b*, c*, diffraction spot distance d*)Structure factor
  • Crystal
  • Crystal lattice (a,b,c, planes distance d)
  • Electron density
  • Fourier Transformhttp://www.nist.gov/lispix/doc/menu-bar/fft-tool.htmTry out an interactive tutorial at:http://www.lks.physik.uni-erlangen.de/diffraction/space.htmlThe Fourier DuckFourier TransformReverseTransformReverseTransformwith limitedresolution dataWhy Use X-rays?http://bccp.lbl.gov/Academy/wksp_pix_1/spectrum.gifX-ray DiffractionGale Rhodes, Crystallography Made Crystal Clear: A Guide for Users of Macromolecular Models, Academic Press, 1993Bragg’s Law
  • n = 2d sin
  • 2 angle between incident
  • and reflected beams
  • d spacing between planes
  • wavelength
  • n order of diffractionhttp://www.bmsc.washington.edu/people/merritt/bc530/bragg/ try the Java Applet!Constructive interference occurs from successive crystallographic planes (h, k, l)in the crystalline lattice Microscopy vs X-ray Crystallographyhttp://www.iucr.org/education/pamphlets/15/full-textThe Phase Problemhttp://www.ruppweb.org/Xray/Phasing/Phasingt.htmlThe X-ray Crystallography Pipeline Crystal growthData collectionPhase determinationModel building and refinementProtein preparationCrystal Growth: Vapor DiffusionCover SlipPrecipitant SolutionProtein + PrecipitantCommon precipitants:
  • Polyethylene glycol
  • Salts
  • ammonium sulfate
  • sodium chloride
  • Alcohols
  • Isopropanol
  • Methylpentanediol (MPD)
  • Data CollectionCrystal mounted in glass capillaryCrystal mounted in nylon loop.Frozen in liquid N2Rotating Anode DiffractometerSynchrotron X-ray sourcehttp://www.nsls.bnl.govNSLS Beamline X12CCrystal DiffractionHigh Resolution(large angle)Water Ring~3-5 ÅBeam Stop ShadowLow Resolution(small angle)Jeff Dahl, Sars protease, http://en.wikipedia.org/wiki/File:X-ray_diffraction_pattern_3clpro.jpgtrp repressor, sodium phosphatetrp repressor, ammonium sulfateDifferent crystal forms of the same protein yield different diffraction patternsX-ray Diffraction Pattern
  • Diffraction pattern is in reciprocal space
  • Size and shape of unit cell determines position of diffraction peaks.
  • Atomic positions within unit cell determines intensity of peaks.
  • Experimental data: h,k,l and intensities (with errors)
  • A precession photographData Obtaineda = 36.67 Å b = 79.39 Å c = 39.97 Å α = 90.0° ß = 91.25° γ = 90.0° Monoclinic lattice (P2 or P21)H K L intensity error 0 0 12 6714.3 347.2 0 0 18 -8.9 16.3 0 0 24 979.5 62.4 0 0 30 4136.4 272.5 1 0 3 3035.4 70.2 1 0 4 0.0 0.7 1 0 5 0.1 0.6 1 0 6 838.4 20.4 1 0 7 14903.0 535.6 1 0 8 2759.4 64.7 1 0 9 1403.5 31.0 1 0 10 109.4 5.6 1 0 11 31739.5 1611.5 1 0 12 231.9 7.6...etc.Crystal unit cell dimensions Lattice type, possible space groupsResolution LimitMerged data set with index, intensity + error for each reflectionIhkl=constant.|Fhkl|2Structure Factor Structure Factor r(x,y,z) =ΣFhkle-2πi (hx + ky +lz)Electron DensityDiffraction Patterns to StructurePhase Problem
  • Structure factor is dependent on type and location of atoms in unit cell
  • The complete Structure Factor Ffor a reflection includes the phase, which cannot be measured directly.
  • Fhkl = |Fhkl|eiϕhklStructure FactorPhase:must be estimatedAmplitude:from experimentalmeasurementsPhase Determination
  • Direct methods
  • Estimate from probability relationships applied to most intense diffraction peaks
  • Patterson methods
  • Multiple Isomorphous Replacement
  • Anomalous Dispersion
  • Molecular replacement
  • Density Improvement
  • Non-crystallographic symmetry averaging
  • Solvent flattening
  • Patterson Function
  • Convolution of electron density with itself
  • Evaluated at points u,v,w throughout unit cell
  • Map of vectors between scattering atom in the real crystal cell (translated to Patterson origin)
  • Patterson mapcrystalhttp://www.ruppweb.org/Xray/Patterson/Native_Patterson.htmIsomorphous Replacement
  • Derivative – native crystal = heavy atom
  • Deriv. diffn – native diffn = heavy atom diffn
  • Patterson synthesis > peaks based on distance between heavy atoms in structure gives initial phase.
  • Real spaceReciprocal spacehttp://www.ruppweb.org/Xray/Phasing/Phasingt.htmlAnomalous Dispersion
  • Friedel’s Law: Ihkl = I-h-k-l
  • Members of a Friedel pair have equal amplitude and opposite phase
  • In anomalous scattering crystals Friedel’s law is not obeyed
  • http://www.xtal.iqfr.csic.es/Cristalografia/parte_07_2-en.htmlhttp://skuld.bmsc.washington.edu/scatter/AS_wavechoice.htmlMolecular Replacement
  • New structure expected to resemble one previously determined
  • Use Patterson-based methods to find the orientation of known model in new crystal lattice (i.e. find rotation R and translation T)
  • http://reference.iucr.org/dictionary/Molecular_replacementElectron Density
  • Can be calculated by Fourier transform of diffraction data
  • Provides an averaged image:
  • over all molecules in the crystal
  • over the time of the diffraction experiment
  • Trp in a 4.3 A mapTrp in a 1.3 A mapTrp in a 2.25 A mapModel Building-Refinement CycleFinal Model-snip-Structural DataPDB 3a6bTypes of Electron Density Maps
  • Experimentally phased map:
  • Fobs, Phicalc
  • “model” map:
  • (2Fobs – Fcalc), Phicalc
  • “difference” map
  • (Fobs – Fcalc) or (Fobs – Fobs), Phicalc
  • R-factor EquationR versus RfreeTypical Statistical TableValidation: Ramachandran PlotSummary: The Data PipelineIsolation, Expression,Purification,CrystallizationGenomicBased Target SelectionDataCollectionStructureDeterminationPDB Deposition & ReleaseX-ray crystNMR3D ModelsAnnotationsPublicationsEMSome Movie Links
  • Crystal Mounting Robot
  • http://www.youtube.com/watch?v=J4OD_b9XKh4
  • Crystal Diffraction
  • http://ucxray.berkeley.edu/~jamesh/movies/
  • Optical diffraction
  • http://mrsec.wisc.edu/Edetc/supplies/DNA_OTK/index.html
  • Enjoy!References
  • IUCr Online dictionary of Crystallography
  • http://reference.iucr.org/dictionary/Main_Page
  • Educational web sites and resources
  • http://www.iucr.org/education/resources
  • An interactive SF tutorial
  • http://www.ysbl.york.ac.uk/~cowtan/sfapplet/sfintro.html
  • Related Search
    We Need Your Support
    Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

    Thanks to everyone for your continued support.

    No, Thanks