BM29 biosaxs data processing tutorial

Transcription

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2 HERCULES 2014 BM29 biosaxs data processing tutorial Page 2

3 OUTLINE Sample Changer Primary Data Processing Model Validation HPLC-SAXS Primary Data Processing Model Validation Ab Initio Model Software in this tutorial: ATSAS by EMBL Hamburg Page 3

4 SAMPLE CHANGER: SCIENTIFIC QUESTION Dimer A or Dimer B Compare theoretical SAXS curve to experiment Page 4

5 SAMPLE CHANGER DATA SET Data set of: protein measurements at 3 different concentrations, 10 frames each 4 buffer measurements, 10 frames each Data are azimuthally integrated Forward scattering of BSA is determined as 14.8 We need to create an idealized curve by comparing and averaging buffer frames comparing and averaging sample frames subtracting buffer data from sample data merging data from different protein concentrations Page 5

6 SAMPLE CHANGER - WORKFLOW 2 x 10 frames bufffer 10 frames sample 2 concentrations pyfai Select Average 1 buffer curve Subtract 1 sample curve Idealized curve 1 curve per concentration dammif damaver dammin datgnom autorg Page 6

7 ISPYB: DATA ANALYSIS OVERVIEW ispyb.esrf.fr Page 7

8 SAMPLE CHANGER - AVERAGING primusqt 8

9 SAMPLE CHANGER - SAMPLE primusqt 9

10 SAMPLE CHANGER - AVERAGING primusqt 10

11 SAMPLE CHANGER - SUBTRACTION primusqt Repeat for every concentration Compare concentrations 11

12 SAMPLE CHANGER IDEALIZED CURVE primusqt Radius of gyration 12

13 SAMPLE CHANGER - IDEALIZED CURVE primusqt Cropped 56 points a beginning Radius of Gyration: 3.2 nm Forward scattering: 10.2 Mass: 10.2/ kda 45 kda 13

14 SAMPLE CHANGER - IDEALIZED CURVE primusqt Change these values 14

15 SAMPLE CHANGER - IDEALIZED CURVE primusqt 15

16 SAMPLE CHANGER - IDEALIZED CURVE primusqt 16

17 SAMPLE CHANGER - IDEALIZED CURVE primusqt Lower limit: autorg Reduce point range Upper limit: noise level 17

18 SAMPLE CHANGER - CRYSOL FIT crysolqw 18

19 SAMPLE CHANGER - CRYSOL FIT crysolqw 19

20 SAMPLE CHANGER - CRYSOL FIT crysolqw Chi: 23 Radius of gyration Model: 3.3 nm Experiment: 3.0 nm Dimer A 20

21 SAMPLE CHANGER - CRYSOL FIT crysolqw Chi: 12.5 Radius of gyration Model: 3.1 nm Experiment: 3.0 nm Dimer B 21

22 SAMPLE CHANGER BEAD MODEL primusqt Example data: Lysozyme 22

23 SAMPLE CHANGER BEAD MODEL primusqt Radius of Gyration: 1.5 nm Forward scattering: 18.5 Mass: 18.5 kda 23

24 SAMPLE CHANGER BEAD MODEL primusqt Dmax = 5.05 Radius of gyration: 1.5 nm Porod Volume: 20.4 nm 3 Mass 20.4/1.7 kda 12 kda 24

25 SAMPLE CHANGER BEAD MODEL dammif Standard parameters are normally fine! 25

26 SAMPLE CHANGER BEAD MODEL dammif Run several times (e.g. 10), then run damaver /a on all models 26

27 SAMPLE CHANGER BEAD MODEL Compare and align models Reject extreme outliers Calculate Intersection (damfilt) Union (damaver) damaver 27

28 SAMPLE CHANGER BEAD MODEL damfilt and damaver do not fit data dammin can calculate a bead model that fits the data is inside the volume of damaver (damaver result damstart) dammin 28

29 OUTLINE Sample Changer Primary Data Processing Model Validation SEC-SAXS Primary Data Processing Model Validation Ab Initio Model Page 29

30 HPLC DATA SET Data set of 1200 frames Data are azimuthally integrated background corrected We need to create an idealized curve by defining a region of interest comparing and averaging sample frames Page 30

31 HPLC- WORKFLOW 1000 frames pyfai Select Average 1 buffer e.g. frame Subtract samples autorg datgnom dammif damaver dammin 1 curve find peak autorg 544 Page 31

32 HPLC DATA ACQUISITION 1000 or more single measurements in a dataset Page 32

33 HPLC SCIENTIFIC CASE solution structure? Problem: Solution also contains aggregates and higher oligomers But these separate nicely on the size exclusion column Page 33

34 HPLC - ISPYB OVERVIEW Run information ispyb.esrf.fr Run overview 34

35 HPLC - ISPYB OVERVIEW ispyb.esrf.fr Select single frame Robust R G Mass should be 45 kda Forward scattering Radius concentration of gyration Mass 16/51 estimate mg/ml 0.3 mg/ml 35

36 HPLC - ISPYB OVERVIEW ispyb.esrf.fr Check buffer & signal 36

37 HPLC - ISPYB OVERVIEW ispyb.esrf.fr Zip file Buffer 1d curves of frames averaged 1d curves of peaks #1 -#4 1d curves corrected for buffer 37

38 HPLC IDEALIZED CURVE primusqt 38

39 HPLC IDEALIZED CURVE primusqt 39

40 HPLC IDEALIZED CURVE primusqt Radius of gyration 40

41 HPLC IDEALIZED CURVE primusqt Cropped 33 points a beginning Radius of Gyration: 3.3 nm 41

42 HPLC IDEALIZED CURVE primusqt Lower limit: autorg Reduce point range Upper limit: noise level 42

43 HPLC - CRYSOL FIT crysolqw 43

44 HPLC - CRYSOL FIT crysolqw Chi

45 HPLC - BEAD MODEL primusqt Distance Distribution 47

46 HPLC - BEAD MODEL primusqt Dmax = Radius of gyration: 3.3 nm Porod Volume: 81 nm 3 Mass 81/1.7 kda 47 kda 48

47 HPLC - BEAD MODEL Run dammif, damaver and dammin as before Page 49

48 TOOLS AND ALTERNATIVES ATSAS (EMBL Hamburg, Includes primus Online version of some tools available ISPYB (ispyb.esrf.fr) Scatter (Robert Rambo, alternative to primus FoXS ( ) - alternative to CRYSOL Page 50