《生物小角散射BioSAXS》由会员分享,可在线阅读,更多相关《生物小角散射BioSAXS(88页珍藏版)》请在金锄头文库上搜索。
1、OutlineSAXS experiment setup3D 2D 1DData reduction and analysisShape and sizeOverall parametersAb initio shape determinationOther methodsAutomationsolutionSmall Angle X-ray Scattering|s| = 4 sin/s scattering vector2 scattering angle wavelengthI(s) intensitySynchrotron Radiation X-ray detector2sHomog
2、eneous andMonodisperse solutionsolventExperiment Setup1-2 mg purified materialconcentration from 0.5 mg/ml, exposure times: a few seconds/minutesX-ray detectorsolutionsolventSynchrotron RadiationSynchrotron Radiation sI(s)Small Angle X-ray ScatteringExperiment Setup1-2 mg purified materialconcentrat
3、ion from 0.5 mg/ml, exposure times: a few seconds/minutesBeambeamstophigher anglelower angleBeamSmall Angle X-ray ScatteringExposureX-ray detectorSmall Angle X-ray ScatteringExposureX-ray detectorNormalization against: data collection time, concentration, transmitted sample intensity.Log I(s),a.u.s,
4、 nm-1|s| = 4 sin/Small Angle X-ray ScatteringRadial averagingBackground subtractionShape and sizelysozymeLog I(s)a.u.s, nm-1Shape and sizelysozymeapoferritinLog I(s)a.u.s, nm-1Crystalsolutionsolutionvs. Thousands of reflectionsThousands of reflections 3D, high resolution3D, high resolution A few Sha
5、nnon channelsA few Shannon channels 1D, low resolution1D, low resolutionCrystalsolutionvs.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 molecula
6、r 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 process
7、esSAXSData processingData qualityRadiation damageLog I(s), a.u.s, nm-1sampleData qualityRadiation damages, nm-1samplesame sample againRADIATION DAMAGE!Log I(s), a.u.Background subtractionsamplesamplebufferLog I(s),a.u.sample buffer(subtracted)Log I(s),a.u.s, nm-1s, nm-1Solution minus SolventLooking
8、for protein signals less than 5% above background levelData quality“Can I use this data for further analysis?”Log I(s)s, nm-1lysozymeLog I(s)s, nm-1AGGREGATED!Data rangeAtomic structureFoldShape051015Log I(s)5678Resolution, nm2.001.000.670.500.33Sizes, nm-1 Dmitri SvergunMerging dataLow and High Con
9、centrationLog I(s)s, nm-1YtvA protein (60 kDa), 1 mg/mlMerging dataLow and High ConcentrationLog I(s)s, nm-11 mg/ml10 mg/mlMerging dataLow and High ConcentrationLog I(s)s, nm-1Merging dataLow and High ConcentrationLog I(s)s, nm-1Merging dataLow and High ConcentrationLog I(s)s, nm-1Merging dataLow an
10、d High ConcentrationLog I(s)s, nm-1Extrapolation to zero concentrationInfinite dilutionLog I(s)s, nm-110 mg/ml1 mg/ml0 mg/ml?SizeLog I(s)s, -1SizeLog I(s)s, -1SizeLog I(s)s, -1Radius of gyration (Rg)DefinitionAverage of square center-of-mass distances in the moleculeweighted by the scattering length
11、 densityMeasure for the overall size of a macromoleculeLog I(s)sGuinier plotRadius of gyration (Rg)Log I(s)sGuinier plotLog I(s)sRadius of gyration (Rg)Ln I(s)s2Guinier plotRadius of gyration (Rg)Ln I(s)s2Guinier plotRadius of gyration (Rg)Log I(s)sNormal Log plotLn I(s)s2Estimate of the overall siz
12、e 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)Ln I(s)s2Estimate of the overall size of the particlesQuality of the dataaggregationpolydispersityimproper background substractionZero angle intensit
13、y 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)s, 1/nmBovine serum albumin (BSA)Log I(s)Radius of gyration (Rg)Ln I(s)s2Guinier plotRadius of gyration (Rg)Ln I(s)s2AUTORGRadius of gyration (Rg)Check all reasonab
14、le linear intervalsFind bestsminRg 1.0smaxRg 1.3Fit qualityGuinier plotLn I(s)s2Guinier plotRadius of gyration (Rg)Ln I(s)s2Guinier plotRadius of Gyration (Rg)y = ax + bRg = sqrt(-3a)Ln I(0)Ln I(s)s2Guinier plotRadius of Gyration (Rg)Rg stdevForward scattering I(0)Data qualityData rangeLn I(0)lysozy
15、meapoferritinLog I(s), a.u.s, nm-1Guinier approximationMolecular massLog I(0)lysLog I(0)apoI(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 kDaBSAI(0
16、) 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 kDaBSAPorod lawI(s) s-4Intensity decay is proportional to s-4 at higher angles (for globular particles
17、of uniform density)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 particlesPorod plot974 nm974 nm3 314 nm14 nm3 3I(s)*s4I(s)*s4ssPrimusExcluded volum
18、e of the hydrated particle9 kDa9 kDa610 kDa610 kDap(r) functionDistance distribution functionr, nmp(r)p(r) functionDistance distribution functionp(r) functionDistance distribution functionIndirect Fourier Transformp(r)p(r)I(s)I(s)p(r) plotDistance distribution functionr, nmr, nmp(r)p(r)GnomDmaxDmaxr
19、, nmp(r)DmaxData qualityI(s)s, 1/nmsminr, nmp(r)DmaxData qualitysmin /DmaxI(s)s, 1/nmsminYeast bleomycin hydrolase 3GCB 50 kDa MonomerCompact dimerExtended dimerHexamerp(r) plotYeast bleomycin hydrolase 3GCB 50 kDa Monomer Compact dimer Extended dimer Hexamerp(r) plot50 kDa Monomer Compact dimer Ext
20、ended dimer HexamerYeast bleomycin hydrolase 3GCB GoodBadSAXS studies of biological macromoleculesRadius of gyrationMolecular massExcluded volumeSAXS studies of biological macromoleculesRadius of gyrationMolecular massExcluded volumeRgMMVolumeAb initio shape determinationDmaxSolventParticleChacn, P.
21、 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 modelAb initio shape determinationDAMMINDis
22、connected Loose CompactP222 symmetryAb initio shape determinationTetrameric pyruvate oxidase from yeast, 240 kDal structure DAMMINTetrameric pyruvate oxidase from yeast Comparison of the ab initio model with the crystal structure Ab initio shape determinationAb initio shape determinationSAXS studies
23、 of biological macromoleculesRgMMVolumeSAXS studies of biological macromoleculesRgMMVolumeShapeValidation in solutionSAXS studies of biological macromoleculesRgMMVolumeShapeRigid body modellingSAXS studies of biological macromoleculesRgMMVolumeShapeRigid body modellingSASREFInterconnectivityAbsence
24、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.
25、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 methodsSAXS studies of
26、biological macromoleculesRgMMVolumeShapeRigid body modellingAdd missing fragmentsSAXS studies of biological macromoleculesRgMMVolumeShapeRigid body modellingMissing fragmentsFlexible systemsOligomeric mixturesSAXS studies of biological macromoleculesRgMMVolumeShapeRigid body modellingMissing fragmen
27、tsFlexible systemsOligomeric mixturesATSASATSASsoftware packagesoftware packageSample preparation ExperimentData processingUnambiguous interpretationProblemsAutomationof the experiment and data analysisAutomated sample changersAt the X33 beamlineExperimental hutchX33 beamlineX33 beamlineRemote contr
28、ol of the experimentRemote control of the experimenthttp:/x33.embl-hamburg.de/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 informationWeb accessWorlds 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 CETThank you!