生物小角散射 BioSAXS.ppt

Hamburg DESY Heidelberg Monterotondo Grenoble ESRF EBI Hinxton EuropeanMolecularBiologyLaboratory AustriaBelgiumCroatiaDenmarkFinlandFranceGermanyGreeceIcelandIrelandIsraelItalyLuxembourgNetherlandsNorwayPortugalSpainSwedenSwitzerlandUnitedKingdom Australia Outline SAXSexperimentsetup3D 2D 1DDatareductionandanalysisShapeandsizeOverallparametersAbinitioshapedeterminationOthermethodsAutomation solution SmallAngleX rayScattering s 4 sin s scatteringvector2 scatteringangle wavelengthI s intensity SynchrotronRadiation X raydetector 2 s HomogeneousandMonodispersesolution solvent ExperimentSetup 1 2mgpurifiedmaterialconcentrationfrom0 5mg ml exposuretimes afewseconds minutes X raydetector solution solvent SmallAngleX rayScattering ExperimentSetup 1 2mgpurifiedmaterialconcentrationfrom0 5mg ml exposuretimes afewseconds minutes SmallAngleX rayScattering Exposure X raydetector SmallAngleX rayScattering Exposure X raydetector SmallAngleX rayScattering Exposure X raydetector Normalizationagainst datacollectiontime concentration transmittedsampleintensity LogI s a u s nm 1 s 4 sin SmallAngleX rayScattering Radialaveraging Backgroundsubtraction Shapeandsize lysozyme LogI s a u s nm 1 Shapeandsize lysozyme apoferritin LogI s a u s nm 1 Crystal solution solution vs Thousandsofreflections3D highresolution AfewShannonchannels1D lowresolution Crystal solution vs Crystal solution vs Noneedtogrowcrystals Nocrystallographicpackingforcesarepresent Notlimitedbymolecularmass Applicableundernearlyanyphysiologicalconditions Observeresponsestochangesinconditions Quantitativeanalysisofcomplexsystemsandprocesses SAXS Dataprocessing Dataquality Radiationdamage LogI s a u s nm 1 sample Dataquality Radiationdamage s nm 1 samplesamesampleagain RADIATIONDAMAGE LogI s a u Backgroundsubtraction sample samplebuffer LogI s a u s nm 1 SolutionminusSolvent Lookingforproteinsignalslessthan5 abovebackgroundlevel Dataquality CanIusethisdataforfurtheranalysis lysozyme Datarange Atomicstructure Fold Shape 0 5 10 15 LogI s 5 6 7 8 Resolution nm 2 00 1 00 0 67 0 50 0 33 Size DmitriSvergun Mergingdata LowandHighConcentration LogI s s nm 1 YtvAprotein 60kDa 1mg ml Mergingdata LowandHighConcentration LogI s s nm 1 1mg ml 10mg ml Mergingdata LowandHighConcentration LogI s s nm 1 Mergingdata LowandHighConcentration LogI s s nm 1 Mergingdata LowandHighConcentration LogI s s nm 1 Mergingdata LowandHighConcentration LogI s s nm 1 Extrapolationtozeroconcentration Infinitedilution LogI s s nm 1 10mg ml 1mg ml 0mg ml Size LogI s s 1 Size LogI s s 1 Size LogI s s 1 Radiusofgyration Rg Definition Averageofsquarecenter of massdistancesinthemoleculeweightedbythescatteringlengthdensity Measurefortheoverallsizeofamacromolecule Guinierplot Radiusofgyration Rg Guinierplot Radiusofgyration Rg Guinierplot Radiusofgyration Rg Guinierplot Radiusofgyration Rg LogI s s NormalLogplot Guinierplot y ax bRg sqrt 3a Andr Guinier1911 2000 Radiusofgyration Rg EstimateoftheoverallsizeoftheparticlesQualityofthedataaggregationpolydispersityimproperbackgroundsubstractionZeroangleintensityI 0 Firstpointtouse Guinierapproximation I s I 0 exp s2Rg2 3 sRg 1 3 Guinierplot y ax bRg sqrt 3a Radiusofgyration Rg s 1 nm Bovineserumalbumin BSA LogI s Radiusofgyration Rg LnI s s2 Guinierplot Radiusofgyration Rg LnI s s2 AUTORG Radiusofgyration Rg Checkallreasonablelinearintervals Findbest sminRg 1 0smaxRg 1 3Fitquality Guinierplot LnI s s2 Guinierplot Radiusofgyration Rg LnI s s2 Guinierplot RadiusofGyration Rg y ax b Rg sqrt 3a LnI 0 LnI s s2 Guinierplot RadiusofGyration Rg Rg stdevForwardscatteringI 0 DataqualityDatarange LnI 0 lysozyme apoferritin LogI s a u s nm 1 Guinierapproximation Molecularmass LogI 0 lys LogI 0 apo RgandI 0 I 0 ZeroAngleIntencity LnI s s2 smin smax LnI 0 Rg 1 46nmI 0 3 66Points63to220smin Rg 0 624 1smax Rg 1 144 1 3 Guinierplot AutoRg I 0 andMolecularMass Rg 1 46nmI 0 3 66MM 20 6kDa MMsample I 0 sample MMBSA I 0 BSA Rg 6 81nmI 0 79 45MM 448 2kDa Rg 3 1nmI 0 11 7MMBSA 66kDa BSA I 0 andMolecularMass Rg 1 46nmI 0 3 66MM 20 6kDa MMsample I 0 sample MMBSA I 0 BSA Rg 6 81nmI 0 79 45MM 448 2kDa Rg 3 1nmI 0 11 7MMBSA 66kDa BSA Porodlaw I s s 4 Intensitydecayisproportionaltos 4athigherangles forglobularparticlesofuniformdensity Porodlaw Excludedvolumeofthehydratedparticle K4isaconstantdeterminedtoensuretheasymptoticalintensitydecayproportionaltos 4athigheranglesfollowingthePorod slawforhomogeneousparticles Porodplot 974nm3 14nm3 I s s4 I s s4 s s Primus Excludedvolumeofthehydratedparticle 9kDa 610kDa p r function Distancedistributionfunction r nm p r p r function Distancedistributionfunction p r function Distancedistributionfunction IndirectFourierTransform p r p r I s I s p r plot Distancedistributionfunction r nm r nm p r p r Gnom r nm p r Dataquality I s s 1 nm smin r nm p r Dataquality smin Dmax I s s 1 nm smin Yeastbleomycinhydrolase3GCB 50kDaMonomer Compactdimer Extendeddimer Hexamer p r plot Yeastbleomycinhydrolase3GCB 50kDaMonomerCompactdimerExtendeddimerHexamer p r plot 50kDaMonomerCompactdimerExtendeddimerHexamer Yeastbleomycinhydrolase3GCB Good Bad SAXSstudiesofbiologicalmacromolecules RadiusofgyrationMolecularmassExcludedvolume SAXSstudiesofbiologicalmacromolecules RadiusofgyrationMolecularmassExcludedvolume RgMMVolume Abinitioshapedetermination Chac n P etal 1998 Biophys J 74 2760 2775Svergun D I 1999 Biophys J 76 2879 2886 Denselypackedbeads Monte Carlotypesearch Findaconfigurationthatyieldsthecalculatedscatteringcurvefittingtheexperimentaldata Abinitioshapedetermination Beadmodel Abinitioshapedetermination MyosinheadsubfragmentS1 experimentaldatafit evolutionofthebeads Abinitioshapedetermination DAMMIN DisconnectedLooseCompact P222symmetry Abinitioshapedetermination Tetramericpyruvateoxidasefromyeast 240kDalstructure DAMMIN TetramericpyruvateoxidasefromyeastComparisonoftheabinitiomodelwiththecrystalstructure Abinitioshapedetermination Abinitioshapedetermination SAXSstudiesofbiologicalmacromolecules RgMMVolume SAXSstudiesofbiologicalmacromolecules RgMMVolume Shape Validationinsolution SAXSstudiesofbiologicalmacromolecules RgMMVolume Shape Rigidbodymodelling SAXSstudiesofbiologicalmacromolecules RgMMVolume Shape Rigidbodymodelling SASREF InterconnectivityAbsenceofstericclashesSymmetryIntersubunitcontacts fromchemicalshiftsbyNMRormutagenesis Distancesbetweenresidues FRETormutagenesis Relativeorientationofsubunits RDCbyNMR Scatteringdatafromsubcomplexes SASREF Petoukhov 2006 Eur Biophys J 35 567 HugeamountofstructuralinformationaboutindividualmacromoleculesLargemacromolecularcomplexesaredifficulttostudybyhighresolutionmethodsHighresolutionmodelsofsubunitscanbeusedtomodelthequaternarystructureofcomplexesbasedonlowresolutionmethods Globalrefinementwithdistanceconstraints AtyrosinekinaseMET 118kDa consistingoffivedomains Gherardi Sandin Petoukhov Finch Youles Ofverstedt Miguel Blundell VandeWoude Skoglund Svergun 2006 PNASUSA 103 4046 Rigidbodymodelling SAXSstudiesofbiologicalmacromolecules RgMMVolume Shape Rigidbodymodelling SAXSstudiesofbiologicalmacromolecules RgMMVolume Shape Rigidbodymodelling Addmissingfragments SAXSstudiesofbiologicalmacromolecules RgMMVolume Shape Rigidbodymodelling Missingfragments Flexiblesystems Oligomericmixtures SAXSstudiesofbiologicalmacromolecules RgMMVolume Shape Rigidbodymodelling Missingfragments Flexiblesystems Oligomericmixtures ATSASsoftwarepackage SamplepreparationExperimentDataprocessingUnambiguousinterpretation Problems Automationoftheexperimentanddataanalysis Automatedsamplechangers AttheX33beamline X33beamline Remotecontroloftheexperiment Remotecontroloftheexperiment http x33 embl hamburg de Datareductionandanalysissteps Radialaveraging Radiationdamagecheck Normalization Buffersubtraction Extrapolationtoinfinitedilution Rg molecularmass Dmax p r volume Abinitioshapedetermination Furtherstepsdependonspecimentypeandavailableaprioriinformation Webaccess World sfirstremoteSAXSexperiment DmitriNanyangTechnologicalUniversitySingapore RemoteaccessinterfacewithcamerasdisplayingthesamplecellandtheSAXSrobot May 26th 2009 12 30CET Nothingknown abinitiolowresolutionstructureCompletehighresolutionstructure