生物小角散射 BioSAXS.ppt

1、Hamburg/DESY ,Heidelberg ,Monterotondo ,Grenoble/ESRF ,EBI/Hinxton ,European Molecular Biology Laboratory,Austria Belgium Croatia Denmark Finland France Germany Greece Iceland Ireland Israel Italy Luxembourg Netherlands Norway Portugal Spain Sweden Switzerland United Kingdom + Australia,a,2,Outline,

2、SAXS experiment setup 3D 2D 1D Data reduction and analysis Shape and size Overall parameters Ab initio shape determination Other methods Automation,a,3,solution,Small Angle X-ray Scattering,|s| = 4 sin/ s scattering vector 2 scattering angle wavelength I(s) intensity,Synchrotron Radiation ,X-ray det

3、ector,2,s,Homogeneous and Monodisperse solution,solvent,Experiment Setup,1-2 mg purified material concentration from 0.5 mg/ml, exposure times: a few seconds/minutes,a,4,X-ray detector,solution,solvent,Small Angle X-ray Scattering,Experiment Setup,1-2 mg purified material concentration from 0.5 mg/m

4、l, exposure times: a few seconds/minutes,a,5,Small Angle X-ray Scattering,Exposure,X-ray detector,a,6,Small Angle X-ray Scattering,Exposure,X-ray detector,a,7,Small Angle X-ray Scattering,Exposure,X-ray detector,a,8,Normalization against: data collection time, concentration, transmitted sample inten

5、sity.,Log I(s), a.u.,s, nm-1,|s| = 4 sin/,Small Angle X-ray Scattering,Radial averaging,Background subtraction,a,9,Shape and size,lysozyme,Log I(s) a.u.,s, nm-1,a,10,Shape and size,lysozyme,apoferritin,Log I(s) a.u.,s, nm-1,a,11,Crystal,solution,solution,vs.,a,12,Thousands of reflections 3D, high re

6、solution,A few Shannon channels 1D, low resolution,Crystal,solution,vs.,a,13,Crystal,solution,vs.,No need to grow crystals,No crystallographic packing forces are present,Not limited by molecular mass,Applicable under nearly any physiological conditions,Observe responses to changes in conditions,Quan

7、titative analysis of complex systems and processes,SAXS,a,14,Data processing,a,15,Data quality,Radiation damage,Log I(s), a.u.,s, nm-1,sample,a,16,Data quality,Radiation damage,s, nm-1,sample same sample again,RADIATION DAMAGE!,Log I(s), a.u.,a,17,Background subtraction,sample,sample buffer,Log I(s)

8、, a.u.,s, nm-1,Solution minus Solvent,Looking for protein signals less than 5% above background level,a,18,Data quality,“Can I use this data for further analysis?”,lysozyme,a,19,Data range,Atomic structure,Fold,Shape,0,5,10,15,Log I(s),5,6,7,8,Resolution, nm,2.00,1.00,0.67,0.50,0.33,Size, Dmitri Sve

9、rgun,a,20,Merging data,Low and High Concentration,Log I(s),s, nm-1,YtvA protein (60 kDa), 1 mg/ml,a,21,Merging data,Low and High Concentration,Log I(s),s, nm-1,1 mg/ml,10 mg/ml,a,22,Merging data,Low and High Concentration,Log I(s),s, nm-1,a,23,Merging data,Low and High Concentration,Log I(s),s, nm-1

10、,a,24,Merging data,Low and High Concentration,Log I(s),s, nm-1,a,25,Merging data,Low and High Concentration,Log I(s),s, nm-1,a,26,Extrapolation to zero concentration,Infinite dilution,Log I(s),s, nm-1,10 mg/ml,1 mg/ml,0 mg/ml?,a,27,Size,Log I(s),s, -1,a,28,Size,Log I(s),s, -1,a,29,Size,Log I(s),s, -

11、1,a,30,Radius of gyration (Rg),Definition,Average of square center-of-mass distances in the molecule weighted by the scattering length density,Measure for the overall size of a macromolecule,a,31,Guinier plot,Radius of gyration (Rg),a,32,Guinier plot,Radius of gyration (Rg),a,33,Guinier plot,Radius

12、of gyration (Rg),a,34,Guinier plot,Radius of gyration (Rg),Log I(s),s,Normal Log plot,a,35,Guinier plot,y = ax + b Rg = sqrt(-3a),Andr Guinier 1911-2000,Radius of gyration (Rg),a,36,Estimate of the overall size of the particles Quality of the data aggregation polydispersity improper background subst

13、raction Zero angle intensity I(0) First point to use,Guinier approximation: I(s) = I(0)exp(-s2Rg2/3) sRg1.3,Guinier plot,y = ax + b Rg = sqrt(-3a),Radius of gyration (Rg),a,37,s, 1/nm,Bovine serum albumin (BSA),Log I(s),Radius of gyration (Rg),a,38,Ln I(s),s2,Guinier plot,Radius of gyration (Rg),a,3

14、9,Ln I(s),s2,AUTORG,Radius of gyration (Rg),Check all reasonable linear intervals,Find best,sminRg 1.0 smaxRg 1.3 Fit quality,Guinier plot,a,40,Ln I(s),s2,Guinier plot,Radius of gyration (Rg),a,41,Ln I(s),s2,Guinier plot,Radius of Gyration (Rg),y = ax + b,Rg = sqrt(-3a),Ln I(0),a,42,Ln I(s),s2,Guini

15、er plot,Radius of Gyration (Rg),Rg stdev Forward scattering I(0) Data quality Data range,Ln I(0),a,43,lysozyme,apoferritin,Log I(s), a.u.,s, nm-1,Guinier approximation,Molecular mass,Log I(0)lys,Log I(0)apo,a,44,Rg and I(0),I(0) - Zero Angle Intencity,Ln I(s),s2,smin,smax,Ln I(0),Rg = 1.46 nm I(0) =

16、 3.66 Points 63 to 220 smin*Rg = 0.624 1 smax*Rg = 1.144 1.3,Guinier plot,AutoRg,a,45,I(0) and Molecular Mass,Rg = 1.46 nm I(0) = 3.66 MM = 20.6 kDa,MMsample = I(0) sample* MMBSA / I(0)BSA,Rg = 6.81 nm I(0) = 79.45 MM = 448.2 kDa,Rg = 3.1 nm I(0) = 11.7 MMBSA = 66 kDa,BSA,a,46,I(0) and Molecular Mas

17、s,Rg = 1.46 nm I(0) = 3.66 MM = 20.6 kDa,MMsample = I(0) sample* MMBSA / I(0)BSA,Rg = 6.81 nm I(0) = 79.45 MM = 448.2 kDa,Rg = 3.1 nm I(0) = 11.7 MMBSA = 66 kDa,BSA,a,47,Porod law,I(s) s-4,Intensity decay is proportional to s-4 at higher angles (for globular particles of uniform density),a,48,Porod

18、law,Excluded volume of the hydrated particle,K4 is a constant determined to ensure the asymptotical intensity decay proportional to s-4 at higher angles following the Porods law for homogeneous particles,a,49,Porod plot,974 nm3,14 nm3,I(s)*s4,I(s)*s4,s,s,Primus,Excluded volume of the hydrated partic

19、le,9 kDa,610 kDa,a,50,p(r) function,Distance distribution function,r, nm,p(r),a,51,p(r) function,Distance distribution function,a,52,p(r) function,Distance distribution function,Indirect Fourier Transform,p(r),p(r),I(s),I(s),a,53,p(r) plot,Distance distribution function,r, nm,r, nm,p(r),p(r),Gnom,a,

20、54,r, nm,p(r),Data quality,I(s),s, 1/nm,smin,a,55,r, nm,p(r),Data quality,smin /Dmax,I(s),s, 1/nm,smin,a,56,Yeast bleomycin hydrolase 3GCB,50 kDa Monomer,Compact dimer,Extended dimer,Hexamer,a,57,p(r) plot,Yeast bleomycin hydrolase 3GCB,50 kDa Monomer Compact dimer Extended dimer Hexamer,a,58,p(r) p

21、lot,50 kDa Monomer Compact dimer Extended dimer Hexamer,Yeast bleomycin hydrolase 3GCB,Good,Bad,a,59,SAXS studies of biological macromolecules,Radius of gyration Molecular mass Excluded volume,a,60,SAXS studies of biological macromolecules,Radius of gyration Molecular mass Excluded volume,Rg MM Volu

22、me,Ab initio shape determination,a,61,Chacn, P. et al. (1998) Biophys. J. 74, 2760-2775 Svergun, D.I. (1999) Biophys. J. 76, 2879-2886,Densely packed beads,Monte-Carlo type search,Find a configuration that yields the calculated scattering curve fitting the experimental data,Ab initio shape determina

23、tion,Bead model,a,62,Ab initio shape determination,Myosin head subfragment S1,experimental data fit,evolution of the beads,a,63,Ab initio shape determination,DAMMIN,Disconnected Loose Compact,a,64,P222 symmetry,Ab initio shape determination,Tetrameric pyruvate oxidase from yeast, 240 kDal structure,

24、DAMMIN,a,65,Tetrameric pyruvate oxidase from yeast Comparison of the ab initio model with the crystal structure,Ab initio shape determination,a,66,Ab initio shape determination,SAXS studies of biological macromolecules,Rg MM Volume,a,67,SAXS studies of biological macromolecules,Rg MM Volume,Shape,Va

25、lidation in solution,a,68,SAXS studies of biological macromolecules,Rg MM Volume,Shape,Rigid body modelling,a,69,SAXS studies of biological macromolecules,Rg MM Volume,Shape,a,70,Rigid body modelling,SASREF,Interconnectivity Absence of steric clashes Symmetry Intersubunit contacts (from chemical shi

26、fts by NMR or mutagenesis) Distances between residues (FRET or mutagenesis) Relative orientation of subunits (RDC by NMR) Scattering data from subcomplexes,SASREF: Petoukhov (2006) Eur. Biophys. J. 35, 567.,Huge amount of structural information about individual macromolecules Large macromolecular co

27、mplexes 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 methods,a,71,Global refinement with distance constraints,A tyrosine kinase MET (118 kDa) consisting of five domains,Gherardi

28、, Sandin, Petoukhov, Finch, Youles, Ofverstedt, Miguel, Blundell, Vande Woude, Skoglund, & Svergun (2006) PNAS USA, 103, 4046.,Rigid body modelling,a,72,SAXS studies of biological macromolecules,Rg MM Volume,Shape,Rigid body modelling,a,73,SAXS studies of biological macromolecules,Rg MM Volume,Shape

29、,Rigid body modelling,Add missing fragments,a,74,SAXS studies of biological macromolecules,Rg MM Volume,Shape,Rigid body modelling,Missing fragments,Flexible systems,Oligomeric mixtures,a,75,SAXS studies of biological macromolecules,Rg MM Volume,Shape,Rigid body modelling,Missing fragments,Flexible

30、systems,Oligomeric mixtures,ATSAS software package,a,76,Sample preparation Experiment Data processing Unambiguous interpretation,Problems,a,77,Automationof the experiment and data analysis,a,78,Automated sample changers,At the X33 beamline,a,79,X33 beamline,a,80,Remote control of the experiment,a,81

31、,Remote control of the experiment,http:/x33.embl-hamburg.de/,a,82,Data reduction and analysis steps,Radial averaging,Radiation damage check,Normalization,Buffer subtraction,Extrapolation to infinite dilution,Rg, molecular mass,Dmax, p(r) , volume,Ab initio shape determination,Further steps depend on

32、 specimen type and available a priori information,a,83,Web access,a,84,Worlds first remote SAXS experiment,Dmitri Nanyang Technological University Singapore,Remote access interface with cameras displaying the sample cell and the SAXS robot,May, 26th, 2009, 12:30 CET,a,85,Nothing known: ab initio low resolution structure Complete hig