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Current Status of BioSAXS Environment
at 23A SWAXS Endstation
Yi-Qi Yeh
NSRRC
2013, Annual Users' Meeting
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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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0
42
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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
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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
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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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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