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

17-24 October 2012 EMBO Course Data reduction and processing tutorial Petr V. Konarev European Molecular Biology Laboratory, Hamburg Outstation BioSAXS group

17-24 October 2012 EMBO Course EMBL BioSAXS beamline X33, 2012 Optics Vacuum cell Completely redesigned 2005-2012 Efficiency gain: about 5-10 times Hutch

17-24 October 2012 EMBO Course PILATUS Pixel X-ray Detector at X33 PILATUS 1M (10*100K modules) Active area 70*420 mm 2 , pixel size: 170 μ m Readout time: 3.6ms, framing rate: 50Hz AgBeh pattern In user operation on X33 from 22.11.07

17-24 October 2012 EMBO Course BIOSAXS beamline P12 (Petra-3) Pilatus 2M Gold nanoparticles Iron oxide nanoparticles Brome mosaic virus First user samples, June 2011

17-24 October 2012 EMBO Course (FIT2DView) View) Pattern (FIT2D 2DPattern Ag-Behenate 2D Ag-Behenate

17-24 October 2012 EMBO Course ASK) (with MASK Pattern (with 2DPattern Ag-Behenate 2D Ag-Behenate

17-24 October 2012 EMBO Course (FIT2DView) View) Pattern (FIT2D 1DPattern Ag-Behenate 1D Ag-Behenate

17-24 October 2012 EMBO Course Channels Creating angular axis Ag-Behenate Angular axis file Makeaxis dialog Axis-adat

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 Hardware- Ab initio modelling independent XML-summary file generation 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 PRIMUS: data processing buffer before sample: BSA 5.4 mg/ml buffer after Plot

17-24 October 2012 EMBO Course PRIMUS: buffer averaging buffer before buffer after buffer averaged Average

17-24 October 2012 EMBO Course PRIMUS: data processing sample: BSA 5.4 mg/ml buffer averaged Plot

17-24 October 2012 EMBO Course PRIMUS: buffer subtraction sample: BSA 5.4 mg/ml buffer averaged subtracted curve Subtract

17-24 October 2012 EMBO Course PRIMUS: data at different angular ranges saxs range waxs range Plot

17-24 October 2012 EMBO Course PRIMUS: merging data saxs range waxs range Merge

17-24 October 2012 EMBO Course PRIMUS: merging data saxs range waxs range merged curve Merge

17-24 October 2012 EMBO Course PRIMUS: Guinier plot 2 Rg = ⋅ − ⋅ 2 I ( s ) I ( 0 ) exp( s 3 ) Rg – radius of gyration Rg = 2.68 +- 1.11e-2 I0 = 271.07 +- 0.605 M ≈ M BSA *(I(0)/I BSA (0)) Guinier

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 ∞ ∫ = π = π − 2 2 2 V 2 I ( 0 ) Q 2 I ( 0 ) s ( I ( s ) K ) ds 0 V – excluded volume of particle V = 92.37 Porod

17-24 October 2012 EMBO Course xtrapolation to zero concentration zero concentration xtrapolation to E E

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

17-24 October 2012 EMBO Course Checking Checking Guinier plot Guinier plot 9.8 mg/ml 1.4 mg/ml 3.8 mg/ml 5.7 mg/ml 8.5 mg/ml

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

17-24 October 2012 EMBO Course Real/ reciprocal Real/ reciprocal space transformation space transformation p(r)=r 2 γ (r) distance distribution function γ 0 (r)= γ (r)/ γ (0) i Probability to find a point at r ij distance r from a given point j inside the particle

17-24 October 2012 EMBO Course Distance distribution function from simple shapes

17-24 October 2012 EMBO Course function of helix helix Distance distribution function of Distance distribution

17-24 October 2012 EMBO Course PRIMUS: GNOM menu Gnom Indirect Fourier Transform Run

17-24 October 2012 EMBO Course PRIMUS: P(R) function Gnom Indirect Fourier Transform

17-24 October 2012 EMBO Course AUTOGNOM – automated version of GNOM for monodisperse systems ♦ In the original version of GNOM the maximum particle size D max is a user-defined parameter and successive calculations with different D max are required to select its optimum value. ♦ This optimum D max should provide a smooth real space distance distribution function p(r) such that p(D max ) and its first derivative p'(D max ) 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 D max 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 D max with GNOM (over-estimation) 12.0 Good fit to experimental data BUT: Distance distribution function p(r) becomes negative

17-24 October 2012 EMBO Course Estimation of D max with GNOM (correct case) 8.0 Good fit to experimental data Distance distribution function p(r) goes smoothly to zero

17-24 October 2012 EMBO Course AUTOGNOM – automated version of GNOM for monodisperse systems ♦ The maximum size is determined from automated comparison of the p(r) functions calculated at different D max values ranging from 2R g to 4R g , where R g is the radius of gyration provided by AUTORG. ♦ 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 D max and the best p(r) function is chosen for the final output. 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