Data reduction and processing tutorial Petr V. Konarev European - - PowerPoint PPT Presentation

Data reduction and processing tutorial

Petr V. Konarev European Molecular Biology Laboratory, Hamburg Outstation BioSAXS group

17-24 October 2012 EMBO Course

Vacuum cell

EMBL BioSAXS beamline X33, 2012

Completely redesigned 2005-2012 Efficiency gain: about 5-10 times Optics Hutch 17-24 October 2012 EMBO Course

PILATUS Pixel X-ray Detector at X33

PILATUS 1M (10*100K modules) Active area 70*420 mm2 , pixel size: 170μm Readout time: 3.6ms, framing rate: 50Hz In user operation on X33 from 22.11.07

AgBeh pattern 17-24 October 2012 EMBO Course

BIOSAXS beamline P12 (Petra-3)

17-24 October 2012 EMBO Course

Gold nanoparticles Iron oxide nanoparticles Brome mosaic virus

Pilatus 2M

First user samples, June 2011

Ag-Behenate Ag-Behenate 2D 2DPattern Pattern (FIT2D (FIT2DView) View)

17-24 October 2012 EMBO Course

Ag-Behenate Ag-Behenate 2D 2DPattern Pattern (with (with MASK ASK)

17-24 October 2012 EMBO Course

Ag-Behenate Ag-Behenate 1D 1DPattern Pattern (FIT2D (FIT2DView) View)

17-24 October 2012 EMBO Course

Creating angular axis

Axis-adat Channels Angular axis file Ag-Behenate Makeaxis dialog

17-24 October 2012 EMBO Course

Raw data reduction steps

• Radial integration of 2D image into 1D curve
• Check for radiation damage, averaging of different time

frames (X33: 8 frames (15 sec), P12: 20 frames (100 ms) )

• Associated errors in the data points are computed from the

numbers of counts using Poisson statistics

• Mask file is used to eliminate beamstop and inactive

detector area

• Exact coordinates of the beam center are required for

integration (determined from AgBeh data)

• Data are normalized to the pindiode value (intensity of the

transmitted beam)

• Data are transferred into ASCII format containing 3

columns: s I(s) Er(s)

17-24 October 2012 EMBO Course

An automated SAXS pipeline at X33 / P12

Data normalization 2D-1D reduction Data processing Check for radiation damage Computation of overall parameters Database search Ab initio modelling XML-summary file generation

Hardware- independent analysis block

17-24 October 2012 EMBO Course

Program PRI MUS- graphical package for data manipulations and analysis

♦data manipulations (averaging, background subtraction, merging of data in

different angular ranges, extrapolation to infinite dilution ) ♦evaluation of radius of gyration and forward intensity (Guinier plot, module AUTORG), estimation of Porod volume ♦calculation of distance/size distribution function p(r)/V(r) (module GNOM) ♦data fitting using the parameters of simple geometrical bodies (ellipsoid, elliptic/hollow cylinder, rectangular prism) (module BODIES) ♦data analysis for polydisperse and interacting systems, mixtures and partially ordered systems (modules OLIGOMER, SVDPLOT, MIXTURE and PEAK)

P.V. Konarev, V.V. Volkov, A.V. Sokolova, M.H.J. Koch, D.I. Svergun J.Appl. Cryst. (2003) 36, 1277-1282 17-24 October 2012 EMBO Course

PRIMUS: graphical user interface

17-24 October 2012 EMBO Course

Plot

PRIMUS: data processing

buffer before sample: BSA 5.4 mg/ml buffer after

17-24 October 2012 EMBO Course

Average

PRIMUS: buffer averaging

buffer before buffer after buffer averaged

17-24 October 2012 EMBO Course

Plot

PRIMUS: data processing

sample: BSA 5.4 mg/ml buffer averaged

17-24 October 2012 EMBO Course

Subtract

PRIMUS: buffer subtraction

sample: BSA 5.4 mg/ml buffer averaged subtracted curve

17-24 October 2012 EMBO Course

Plot

PRIMUS: data at different angular ranges

saxs range waxs range

17-24 October 2012 EMBO Course

Merge

PRIMUS: merging data

saxs range waxs range

17-24 October 2012 EMBO Course

Merge

PRIMUS: merging data

saxs range waxs range merged curve

17-24 October 2012 EMBO Course

Guinier

PRIMUS: Guinier plot

Rg = 2.68 +- 1.11e-2 I0 = 271.07 +- 0.605

) 3 exp( ) ( ) (

2 2 Rg

s I s I ⋅ − ⋅ =

Rg – radius of gyration

M≈MBSA*(I(0)/IBSA(0))

17-24 October 2012 EMBO Course

PRIMUS: AutoRG module

AutoRg

Petoukhov, M.V., Konarev, P.V., Kikhney, A.G. & Svergun, D.I. (2007) J. Appl. Cryst., 40, s223-s228. 17-24 October 2012 EMBO Course

PRIMUS: Porod volume

Porod

) ) ( ( ) ( 2 ) ( 2

2 2 2

∫

∞

− = = ds K s I s I Q I V π π

V – excluded volume of particle

V = 92.37

17-24 October 2012 EMBO Course

E xtrapolation to E xtrapolation to zero concentration zero concentration

17-24 October 2012 EMBO Course

Merging for making Merging for making final composite cur final composite curve ve

9.3 mg/ml 1.4 mg/m 3.8 mg/ml 5.7 mg/ml 8.5 mg/mll 9.3 mg/ml 1.4 mg/m 3.8 mg/ml 5.7 mg/ml 8.5 mg/mll

17-24 October 2012 EMBO Course

9.8 mg/ml

Checking Checking Guinier plot Guinier plot

1.4 mg/ml 3.8 mg/ml 5.7 mg/ml 8.5 mg/ml

17-24 October 2012 EMBO Course

Merging Merging for making for making final composite cur final composite curve ve

9.3 mg/ml 1.4 mg/m 3.8 mg/ml 5.7 mg/ml 8.5 mg/mll 9.3 mg/ml 1.4 mg/m 3.8 mg/ml 5.7 mg/ml 8.5 mg/mll 9.3 mg/ml 1.4 mg/m 3.8 mg/ml 5.7 mg/ml 8.5 mg/mll 9.3 mg/ml 1.4 mg/m 3.8 mg/ml 5.7 mg/ml 8.5 mg/mll merged

17-24 October 2012 EMBO Course

Real/ reciprocal Real/ reciprocal space transformation space transformation

p(r)=r2 γ(r) distance distribution function γ0(r)=γ(r)/γ(0)

Probability to find a point at distance r from a given point inside the particle

i

j

rij

17-24 October 2012 EMBO Course

Distance distribution function from simple shapes

17-24 October 2012 EMBO Course

Distance distribution Distance distribution function of function of helix helix

17-24 October 2012 EMBO Course

Gnom Run

PRIMUS: GNOM menu

Indirect Fourier Transform

17-24 October 2012 EMBO Course

Gnom

PRIMUS: P(R) function

Indirect Fourier Transform

17-24 October 2012 EMBO Course

♦ In the original version of GNOM the maximum particle

size Dmax is a user-defined parameter and successive calculations with different Dmax are required to select its

• ptimum value.

AUTOGNOM – automated version of GNOM for monodisperse systems

♦ This optimum Dmax should provide a smooth real

space distance distribution function p(r) such that p(Dmax) and its first derivative p'(Dmax) are approaching zero, and the back-transformed intensity from the p(r) fits the experimental data.

Petoukhov, M.V., Konarev, P.V., Kikhney, A.G. & Svergun, D.I. (2007) J. Appl. Cryst., 40, s223-s228. 17-24 October 2012 EMBO Course

Estimation of Dmax with GNOM (under-estimation)

6.0

Poor fit to experimental data Distance distribution function p(r) goes to zero too abruptly

17-24 October 2012 EMBO Course

Estimation of Dmax with GNOM (over-estimation) Good fit to experimental data BUT: Distance distribution function p(r) becomes negative

12.0

17-24 October 2012 EMBO Course

8.0

Estimation of Dmax with GNOM (correct case) Good fit to experimental data Distance distribution function p(r) goes smoothly to zero

17-24 October 2012 EMBO Course

♦ The maximum size is determined from automated

comparison of the p(r) functions calculated at different Dmax values ranging from 2Rg to 4Rg, where Rg is the radius of gyration provided by AUTORG.

AUTOGNOM – automated version of GNOM for monodisperse systems

♦The calculated p(r) functions and corresponding fits to

the experimental curves are compared using the perceptual criteria of GNOM (Svergun, 1992) together with the analysis of the behavior of p(r) function near Dmax and the best p(r) function is chosen for the final

• utput.

Petoukhov, M.V., Konarev, P.V., Kikhney, A.G. & Svergun, D.I. (2007) J. Appl. Cryst., 40, s223-s228. 17-24 October 2012 EMBO Course

Examples for data processing

Go to directory /Examples/Primus/ ../AgBeh - data from saxs and waxs range as well as the merged curve ../BSA - standard sample for calibration of molecular mass ../Lysozyme – example of p(r) from globular particle ../Lymazine – example of p(r) from hollow globular (virus-like) particle ../ISWI_Cterm – example of p(r) from elongated particle 17-24 October 2012 EMBO Course