生物小角散射 课件

1、Hamburg/DESY,Heidelberg,Monterotondo,Grenoble/ESRF,EBI/Hinxton,EuropeanMolecularBiologyLaboratory,AustriaBelgiumCroatiaDenmarkFinlandFranceGermanyGreeceIcelandIrelandIsraelItalyLuxembourgNetherlandsNorwayPortugalSpainSwedenSwitzerlandUnitedKingdom+Australia,Outline,SAXSexperimentsetup3D2D1DDatareduc

2、tionandanalysisShapeandsizeOverallparametersAbinitioshapedeterminationOthermethodsAutomation,solution,SmallAngleX-rayScattering,|s|=4sin/sscatteringvector2scatteringanglewavelengthI(s)intensity,SynchrotronRadiation,X-raydetector,2,s,HomogeneousandMonodispersesolution,solvent,ExperimentSetup,1-2mgpur

3、ifiedmaterialconcentrationfrom0.5mg/ml,exposuretimes:afewseconds/minutes,X-raydetector,solution,solvent,SmallAngleX-rayScattering,ExperimentSetup,1-2mgpurifiedmaterialconcentrationfrom0.5mg/ml,exposuretimes:afewseconds/minutes,SmallAngleX-rayScattering,Exposure,X-raydetector,SmallAngleX-rayScatterin

4、g,Exposure,X-raydetector,SmallAngleX-rayScattering,Exposure,X-raydetector,Normalizationagainst:datacollectiontime,concentration,transmittedsampleintensity.,LogI(s),a.u.,s,nm-1,|s|=4sin/,SmallAngleX-rayScattering,Radialaveraging,Backgroundsubtraction,Shapeandsize,lysozyme,LogI(s)a.u.,s,nm-1,Shapeands

5、ize,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,Applicableundernear

6、lyanyphysiologicalconditions,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,

7、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,Mergi

8、ngdata,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,Mergingda

9、ta,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-massdistancesinthemoleculeweightedbythescatteringlengthdens

10、ity,Measurefortheoverallsizeofamacromolecule,Guinierplot,Radiusofgyration(Rg),Guinierplot,Radiusofgyration(Rg),Guinierplot,Radiusofgyration(Rg),Guinierplot,Radiusofgyration(Rg),LogI(s),s,NormalLogplot,Guinierplot,y=ax+bRg=sqrt(-3a),AndrGuinier1911-2000,Radiusofgyration(Rg),Estimateoftheoverallsizeof

11、theparticlesQualityofthedataaggregationpolydispersityimproperbackgroundsubstractionZeroangleintensityI(0)Firstpointtouse,Guinierapproximation:I(s)=I(0)exp(-s2Rg2/3)sRg1.3,Guinierplot,y=ax+bRg=sqrt(-3a),Radiusofgyration(Rg),s,1/nm,Bovineserumalbumin(BSA),LogI(s),Radiusofgyration(Rg),LnI(s),s2,Guinier

12、plot,Radiusofgyration(Rg),LnI(s),s2,AUTORG,Radiusofgyration(Rg),Checkallreasonablelinearintervals,Findbest,sminRg1.0smaxRg1.3Fitquality,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),RgstdevForwardscatteringI(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).,Scatteringf