BioSAXS current status - nsrrc.org.t · 50 2005 2006 2007 2008 2009 2010 2011 2012 ... • Check samples for radiation damage ... (BioSAXS check list) 11 ATSAS 2.5.1 (analysis software)

Current Status of BioSAXS Environment

at 23A SWAXS Endstation

Yi-Qi Yeh

NSRRC

2013, Annual Users' Meeting

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2

Outline

� Instrument, Sample preparation environment

Data Processing, Evaluation, and Simulation

1. Data collection and evaluation• Check samples for radiation damage

• Identify the concentration dependence

2. Data merge • Identify the potential interference-free q-region

• Data Merge by Primus

3 Data analysis and Model simulation• Transform data to p(r) with GNOM program and data fitting.

• Crysol fitting --- Compare (fit) solution structure with PDB crystal structure.

• Dammin / Gasbor Program --- An ensemble of dummy atom/residues model

simulation with P(r) output by GNOM.

• SASref --- quaternary structure modeling of a complex formed by subunits

Detecting System

MarCCD (SAXS)

Pilatus 1M-F (SAXS)

Flat Panel (2D WAXS)

1D Gas Detector

(WAXS-H)

Mythen 3K

(WAXS-V)

BL23A instrument setup

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instrument

~

1

28

34

0

42

50

0 0 1 0 1

4

1 20 0 0 0 0 0 1 00 1 0 0 1 0

2 20 1 1 0 0 0 1 0

0

5

10

15

20

25

30

35

40

45

50

2005 2006 2007 2008 2009 2010 2011 2012

Taiwan

Singapore

Korea

Japan

Australia

USA

Year

Nu

mb

er o

f Use

r Gro

up

Biomacromol.35%

polymer35%

Nanocomposites15%

Colloid. Sol.15%Others

10%

Output 2005 (01B) 2006(01B) 2007(17B3) 2008 (17B3) 2009(17B3) 2010(17B3) 2011 (23A) 2012 (23A)

SCI papers 6 6 17 9 20 11 19 37

Average I.F. value

3.705 3.414 4.230 4.938 4.919 4.176 6.116 4.950

23A SWAXS endstation current status, Since May 201050-60 proposals per cycle submitted for beamtimes

23A SWAXS user distribution

Saturated

(2006-2008 17B3 SWAXS endstation, shutdown in 2008-2009)

1. Simultaneous SAXS/WAXS with differential scanning calorimetry (DSC), shearing, drawing, temp.-jump, stopped-flow device, UV-vis absorption, etc.,

2. 10 ms-resolved SAXS/WAXS3. Anomalous SAXS for protein solution and multi-element nanoparticles 4. Time-resolved/anomalous GISAXS/GIWAXS for oriented lipid/peptide membranes,

polymer thin films, and structural evolution at the air-water/liquid-liquid interfaces

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Centrifuge

Cell cleaning

Refrigeratoroff-line HPLC

At BL23A Endstation At Room E171

1. 4~10°C refrigerator: for stand-by samples/sample cell

2. Low temp. Enpendorf Centrifuge (max: 15000 rpm)

3. Off-line HPLC variable UV system (with temp.

Controlled column down to 4 oC)

4. Ultrasonic water injection system/Waste drawing

system: for quick cell cleaning

BioSAXS sample preparation environment

0.1

0.000

0.002

0.004

0.006

0.008

Cytc-10mg/ml Cytc-5mg/ml Cytc-2mg/ml

I(q)

/c (

cm-1m

g-1m

l)

q (ÅÅÅÅ-1)

1. Data collection and evaluation

• Check samples for radiation damage with multi-frame data collection mode (Pilatus 1M-F)

0.02 0.03 0.04 0.05 0.06 0.07 0.080.02

0.04

0.06

0.08

0.1 1st frame 2nd frame 3rd frame 4th frame 5th frame 6th frame combined

I(q)

(cm

-1)

q (Å -1)

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Well overlapped

Radiation damageAggregationInter-particle interference

low-q behavior:

• Identify the concentration dependence

Interparticle interaction

0.01 0.1

0.00

0.03

0.06

0.09

0.12

q (ÅÅÅÅ-1)

I(q)

(cm

-1)

PmrA-1mg/ml PmrA-0.2mg/ml

Aggregation

0.01 0.1

1E-4

1E-3

0.01

0.1

q (ÅÅÅÅ-1)

I(q)

(cm

-1)

PmrA-1mg/ml PmrA-0.2mg/ml

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• Identify the potential interference-free q-region for data merging.

Data from Dr. CD Hsiao

• Identify the potential aggregation

� Primus program

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Merging points

2. Data merge

0.11E-5

1E-4

1E-3

0.01

merged profile PmrA

I(q)

(cm

-1)

q (Å-1)

• GNOM ProgramTransform data to distance distribution function p(r) with GNOM program and do data fit.

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Dmitri I Svergun and Michel H J Koch, EMBL Rep. Prog. Phys. 66 (2003) 1735

Distance distribution functions of typical geometrical bodies.PmrA, Dmax = 105 Å

Available on-site DATA simulation kit and instruction (BioSAXS check list)

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ATSAS 2.5.1 (analysis software)

VMD (molecular graphics software)

With PDB file

BL23AOn-site DATA

Simulation

• Open the log file to read the experimental and theoretical Rg values and 7.08

� Crysol fittingCompare (fit) solution structure with PDB crystal structure.

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3. Data analysis and Model simulation

�Dammin ProgramAn ensemble of dummy atom model simulation with P(r) output by GNOM.

Final SQRT(Chi) against raw data: 1.069

GASBOR Output Files:

1. log file: contain the same information as the screen output.

2. fir file: fit to the raw experimental data

3. pdb file: resulting model in PDB-like format that can be viewed with MASSHA (in ATSAS package) or VMD

(molecular visualization program)

Displayed by VMD

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Dammin

�Gasbor ProgramAn ensemble of dummy residues model simulation with P(r) output by GNOM.

Final Chi2 against raw data .......... : 14.508Final Chi2 against corrected data . : 1.874

GASBOR Output Files:

1. log file: contain the same information as the screen output.

2. fir file: fit to the raw experimental data

3. pdb file: resulting model in PDB-like format that can be viewed with MASSHA (in ATSAS package) or VMD

(molecular visualization program)

Simulation by Gasbor

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Gasbor

PDB structure

0.01 0.110-4

10-3

10-2

10-1

CBP21-(EAAAK)5-NCTU2 (pH9) iEXP Sasref (χχχχ=2.69)

I(q)

(cm

-1)

q (Å-1)

5.65 nm

3.19 nm

CBP21

NCTU2

pH =9

Fitting SAXS data with SASref for relative orientation of two-domain proteins of respective PDB files (CY Chen et al., NSRRC)

Protein Solution SAXS

� Calculate I(0) in absolute intensity scale

protein SAXS I0 estimtaion.xls. (spreadsheet edited by Dr. Jeng)

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Fill in protein conc. & aggregation #Input measured I(0)

Fill in protein amino acids numbers

I(0)

oligomerizationstatus

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Data processing

Data analysis and simulation

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Acknowledge EMBL Hamburg BioSAXS team memebers’ effort in ATSAS

software package. The data analysis software are downloaded from

http://www.embl-hamburg.de/biosaxs/atsas-online/download.php

If one use some programs in the work, please cite the corresponding papers

If user has software problem , please connect the user forum

http://www.saxier.org/forum/viewforum.php?f=5

Other possible packages: FoxS, PyMol, and etc.

Acknowledge

Dr. U-Ser Jeng, NSRRC

Dr. Chun-Jen Su, NSRRC

Dr. Chun-Yu Chen, NSRRC

Ms. Kuei-Fen Liao, NSRRC

Mr. Wei-Ru Wu, NSRRC

Mr. Wen-Bin Su, NSRRC

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This file is available at the NSRRC 23A SWAXS website