生物小角散射-BioSAXS.ppt

OutlineSAXS experiment setup3D 2D 1DData reduction and analysisShape and sizeOverall parametersAb initio shape determinationOther methodsAutomation2.solutionSmall Angle X-ray Scattering|s|=4 sin/s scattering vector2 scattering angle wavelengthI(s)intensitySynchrotron Radiation X-ray detector2sHomogeneous andMonodisperse solutionsolventExperiment Setup1-2 mg purified materialconcentration from 0.5 mg/ml,exposure times:a few seconds/minutes3.X-ray detectorsolutionsolventSynchrotron RadiationSynchrotron Radiation sI(s)Small Angle X-ray ScatteringExperiment Setup1-2 mg purified materialconcentration from 0.5 mg/ml,exposure times:a few seconds/minutes4.Beambeamstophigher anglelower angleBeamSmall Angle X-ray ScatteringExposureX-ray detector5.Small Angle X-ray ScatteringExposureX-ray detector6.Normalization against:data collection time,concentration,transmitted sample intensity.Log I(s),a.u.s,nm-1|s|=4 sin/Small Angle X-ray ScatteringRadial averagingBackground subtraction8.Shape and sizelysozymeLog I(s)a.u.s,nm-19.Shape and sizelysozymeapoferritinLog I(s)a.u.s,nm-110.Crystalsolutionsolutionvs.11.Thousands of reflectionsThousands of reflections 3D,high resolution3D,high resolution A few Shannon channelsA few Shannon channels 1D,low resolution1D,low resolutionCrystalsolutionvs.12.Crystalsolutionvs.No need to grow crystalsNo need to grow crystalsNo crystallographic packing No crystallographic packing forces are presentforces are presentNot limited by molecular massNot limited by molecular massApplicable under nearly any Applicable under nearly any physiological conditionsphysiological conditionsObserve responses to changes Observe responses to changes in conditionsin conditionsQuantitative analysis of complex Quantitative analysis of complex systems and processessystems and processesSAXS13.Data processing14.Data qualityRadiation damageLog I(s),a.u.s,nm-1sample15.Data qualityRadiation damages,nm-1samplesame sample againRADIATION DAMAGE!Log I(s),a.u.16.Background subtractionsamplesamplebufferLog I(s),a.u.sample buffer(subtracted)Log I(s),a.u.s,nm-1s,nm-1Solution minus SolventLooking for protein signals less than 5%above background level17.Data quality“Can I use this data for further analysis?”Log I(s)s,nm-1lysozymeLog I(s)s,nm-1AGGREGATED!18.Data rangeAtomic structureFoldShape051015Log I(s)5678Resolution,nm2.001.000.670.500.33Sizes,nm-1 Dmitri Svergun19.Merging dataLow and High ConcentrationLog I(s)s,nm-1YtvA protein(60 kDa),1 mg/ml20.Merging dataLow and High ConcentrationLog I(s)s,nm-11 mg/ml10 mg/ml21.Merging dataLow and High ConcentrationLog I(s)s,nm-122.Merging dataLow and High ConcentrationLog I(s)s,nm-123.Merging dataLow and High ConcentrationLog I(s)s,nm-124.Merging dataLow and High ConcentrationLog I(s)s,nm-125.Extrapolation to zero concentrationInfinite dilutionLog I(s)s,nm-110 mg/ml1 mg/ml0 mg/ml?26.SizeLog I(s)s,-127.SizeLog I(s)s,-128.SizeLog I(s)s,-129.Radius of gyration(Rg)DefinitionAverage of square center-of-mass distances in the moleculeweighted by the scattering length densityMeasure for the overall size of a macromolecule30.Log I(s)sGuinier plotRadius of gyration(Rg)31.Log I(s)sGuinier plotLog I(s)sRadius of gyration(Rg)32.Ln I(s)s2Guinier plotRadius of gyration(Rg)33.Ln I(s)s2Guinier plotRadius of gyration(Rg)Log I(s)sNormal Log plot34.Ln I(s)s2Estimate of the overall size of the particlesGuinier approximation:I(s)=I(0)exp(-s2Rg2/3)sRg1.3Guinier ploty=ax+b Rg=sqrt(-3a)Andr Guinier1911-2000Radius of gyration(Rg)35.Ln I(s)s2Estimate of the overall size of the particlesQuality of the dataaggregationpolydispersityimproper background substractionZero angle intensity I(0)First point to useGuinier approximation:I(s)=I(0)exp(-s2Rg2/3)sRg1.3Guinier ploty=ax+b Rg=sqrt(-3a)Radius of gyration(Rg)36.s,1/nmBovine serum albumin(BSA)Log I(s)Radius of gyration(Rg)37.Ln I(s)s2Guinier plotRadius of gyration(Rg)38.Ln I(s)s2AUTORGRadius of gyration(Rg)Check all reasonable linear intervalsFind bestsminRg 1.0smaxRg 1.3Fit qualityGuinier plot39.Ln I(s)s2Guinier plotRadius of gyration(Rg)40.Ln I(s)s2Guinier plotRadius of Gyration(Rg)y=ax+bRg=sqrt(-3a)Ln I(0)41.Ln I(s)s2Guinier plotRadius of Gyration(Rg)Rg stdevForward scattering I(0)Data qualityData rangeLn I(0)42.lysozymeapoferritinLog I(s),a.u.s,nm-1Guinier approximationMolecular massLog I(0)lysLog I(0)apo43.I(0)and Molecular MassMMsampleMMBSAI(0)sampleI(0)BSA=Rg=1.46 nmI(0)=3.66MM=20.6 kDaMMsample=I(0)sample*MMBSA/I(0)BSARg=6.81 nmI(0)=79.45MM=448.2 kDaRg=3.1 nmI(0)=11.7MMBSA=66 kDaBSA45.I(0)and Molecular MassMMsampleMMBSAI(0)sampleI(0)BSA=Rg=1.46 nmI(0)=3.66MM=20.6 kDaMMsample=I(0)sample*MMBSA/I(0)BSARg=6.81 nmI(0)=79.45MM=448.2 kDaRg=3.1 nmI(0)=11.7MMBSA=66 kDaBSA46.Porod lawI(s)s-4Intensity decay is proportional to s-4 at higher angles(for globular particles of uniform density)47.Porod lawExcluded volume of the hydrated particleK4 is a constant determined to ensure the asymptotical intensity decay proportional to s-4 at higher angles following the Porods law for homogeneous particles48.Porod plot974 nm974 nm3 314 nm14 nm3 3I(s)*s4I(s)*s4ssPrimusExcluded volume of the hydrated particle9 kDa9 kDa610 kDa610 kDa49.p(r)functionDistance distribution functionr,nmp(r)50.p(r)functionDistance distribution function51.p(r)functionDistance distribution functionIndirect Fourier Transformp(r)p(r)I(s)I(s)52.p(r)plotDistance distribution functionr,nmr,nmp(r)p(r)GnomDmaxDmax53.r,nmp(r)DmaxData qualityI(s)s,1/nmsmin54.r,nmp(r)DmaxData qualitysmin /DmaxI(s)s,1/nmsmin55.Yeast bleomycin hydrolase 3GCB 50 kDa MonomerCompact dimerExtended dimerHexamer56.p(r)plotYeast bleomycin hydrolase 3GCB 50 kDa Monomer Compact dimer Extended dimer Hexamer57.p(r)plot50 kDa Monomer Compact dimer Extended dimer HexamerYeast bleomycin hydrolase 3GCB GoodBad58.SAXS studies of biological macromoleculesRadius of gyrationMolecular massExcluded volume59.SAXS studies of biological macromoleculesRadius of gyrationMolecular massExcluded volumeRgMMVolumeAb initio shape determination60.DmaxSolventParticleChacn,P.et al.(1998)Biophys.J.74,2760-2775Svergun,D.I.(1999)Biophys.J.76,2879-2886Densely packed beadsMonte-Carlo type searchFind a configuration that yields the calculated scattering curve fitting the experimental dataAb initio shape determinationBead model61.Ab initio shape determinationDAMMINDisconnected Loose Compact63.P222 symmetryAb initio shape determinationTetrameric pyruvate oxidase from yeast,240 kDal structure DAMMIN64.Tetrameric pyruvate oxidase from yeast Comparison of the ab initio model with the crystal structure Ab initio shape determination65.Ab initio shape determinationSAXS studies of biological macromoleculesRgMMVolume66.SAXS studies of biological macromoleculesRgMMVolumeShapeValidation in solution67.SAXS studies of biological macromoleculesRgMMVolumeShapeRigid body modelling68.SAXS studies of biological macromoleculesRgMMVolumeShape69.Rigid body modellingSASREFInterconnectivityAbsence of steric clashes SymmetryIntersubunit contacts(from chemical shifts by NMR or mutagenesis)Distances between residues(FRET or mutagenesis)Relative orientation of subunits(RDC by NMR)Scattering data from subcomplexesSASREF:Petoukhov&Svergun(2005)Biophys J.89,1237;(2006)Eur.Biophys.J.35,567.Huge amount of structural information about individual macromolecules Large macromolecular complexes are difficult to study by high resolution methods High resolution models of subunits can be used to model the quaternary structure of complexes based on low resolution methods70.SAXS studies of biological macromoleculesRgMMVolumeShapeRigid body modellingAdd missing fragments73.SAXS studies of biological macromoleculesRgMMVolumeShapeRigid body modellingMissing fragmentsFlexible systemsOligomeric mixtures74.SAXS studies of biological macromoleculesRgMMVolumeShapeRigid body modellingMissing fragmentsFlexible systemsOligomeric mixturesATSASATSASsoftware packagesoftware package75.Sample preparation ExperimentData processingUnambiguous interpretationProblems76.Automationof the experiment and data analysis77.Automated sample changersAt the X33 beamlineExperimental hutch78.X33 beamlineX33 beamline79.Remote control of the experiment80.Remote control of the experimenthttp:/x33.embl-hamburg.de/81.Data reduction and analysis stepsRadial averagingRadiation damage checkNormalizationBuffer subtractionExtrapolation to infinite dilutionRg,molecular massDmax,p(r),volumeAb initio shape determinationFurther steps depend on specimen type and available a priori information82.Web access83.Worlds first remote SAXS experimentEMBL-Hamburgvia SkypeDmitriNanyang Technological University SingaporeRemote access interface with cameras displaying the sample cell and the SAXS robotMay,26th,2009,12:30 CET84.Thank you!86.