Methods for Structure Determination

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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.
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 collectionPhasing 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. Jaskolskib International TablesDiffractionSunrise through a screened window in Action 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 Transform out an interactive tutorial at: Fourier DuckFourier TransformReverseTransformReverseTransformwith limitedresolution dataWhy Use X-rays? 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 diffraction try the Java Applet!Constructive interference occurs from successive crystallographic planes (h, k, l)in the crystalline lattice Microscopy vs X-ray Crystallography Phase Problem 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 source Beamline X12CCrystal DiffractionHigh Resolution(large angle)Water Ring~3-5 ÅBeam Stop ShadowLow Resolution(small angle)Jeff Dahl, Sars protease, 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 mapcrystal 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 space 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
  • 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)
  • 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
  • Crystal Diffraction
  • Optical diffraction
  • Enjoy!References
  • IUCr Online dictionary of Crystallography
  • Educational web sites and resources
  • An interactive SF tutorial
  • Related Search
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