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Solution SAXS in Grenoble
What can be done and how to do it
Adam Round
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• What is Solution SAS
• What can you learn from solution SAS
• Basics of what can be obtained
• Analytical analysis
• Modelling
• Examples
• How to collect data
• Automation
• Data collection and analysis
Contents
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Solution scattering data collection
X-ray detector
Sample
X-ray Beam
X-ray Scattering
sample sample
buffer sample – buffer
(subtracted)
Log I(s)
s, nm-1
Thanks to A. Kikhney for this slide
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Experimental Equipment: The Basics
Source of Radiation Neutrons / X-rays
Detector Sample Holder
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Calibration
All pixel positions with
equal 2θ are averaged
θconverted to s based on known beamline parameters
for geometry and λ
Data are scaled for intensity Based on measurement of
intensity of direct beam
Conversion 2D to 1D
Performed automatically by Beamline softwarebasedoncalibrationbyLC’s
2θ
2θ
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Calibration: Y
Intensity is proportional to: The number of interactions between
Scattered particles (Neutrons/X-rays)
Scattering object (Biological Macromolecule)
And
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Calibration: Y
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Calibration: Y
12 20 6 20
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Calibration: Y
12 20 6 10
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Calibration: Y
12 20 12 20
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Calibration
Scattering intensity
Is proportional to: The number of interactions between
Scattered particles (Neutrons/X-rays)
and
Scattering object (Biological Macromolecule)
Therefore if:
Neutron/X-ray flux is normalised
And
The observed scattering intensity is corrected for the number of scatterers
(proportional to concentration)
The resulting measured intensity is proportional to the size and contrast
of the scattering particles under investigation
Warning! Concentration measurements might not be accurate
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Intensity Calibration
Absolute Relitive
Reference Water Known protein
BSA or Lysozyme
Source of
Error
Protein
Concentration
Protein and reference sample
Concentration
Done by Local Contact User
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Radius of Gyration and Zero Angle Intensity
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Porod Analysis in Primus gives excluded
volume
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Indirect Fourier Transform!
Distance Distribution P(r) Function Calculated by GNOM give the Dmax and the input file required for Ab-initio modeling
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Indirect Fourier Transform!
1D curve and P(r) Function are effected by shape
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Model independent parameters:
Size! Rg, Dmax, Volume and MM estimates. Basic shape (Extended or Globular)
Behaviour in different buffer conditions:
To assess
effects of different buffer formulations
if interparticle scattering which shows structural effects (find optimum conditions – monitor behaviour)
Dynamic investigations under physiological conditions: Conformational changes with temperature, pH, binding etc.
What can we learn from solution SAXS
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Form Factor fitting of basic shapes
1. Homogeneous sphere
2. Spherical shell
3. Spherical concentric shells
4. Particles consisting of spherical subunits
5. Ellipsoid of revolution
6. Tri-axial ellipsoid
7. Cube and rectangular parallelepipedons
8. Truncated octahedra
9. Faceted Sphere
10. Cube with terraces
11. Cylinder
12. Cylinder with elliptical cross section
13. Toroid
14. Infinitely thin rod
15. Infinitely thin circular disk
Adapted from talk of JS Pederson
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Form Factor fitting of polymers
1. Flexible polymers with Gaussian statistics
2. Polydisperse flexible polymers with Gaussian statistics
3. Flexible ring polymers with Gaussian statistics
4. Flexible self-avoiding polymers
5. Polydisperse flexible self-avoiding polymers
6. Semi-flexible polymers without self-avoidance
7. Semi-flexible polymers with self-avoidance:
8. Polyelectrolyte Semi-flexible polymers with self-avoidance:
9. Star polymer with Gaussian statistics
10. Polydisperse star polymer with Gaussian statistics
11. Regular star-burst polymer (dendrimer) with Gaussian statistics
12. Regular comb polymer with Gaussian statistics
13. Arbitrarily branched polymers with Gaussian statistics
14. Arbitrarily branched semi-flexible polymers
15. Arbitrarily branched self-avoiding polymers
Adapted from talk of JS Pederson
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Combining Form Factors
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Self-assembling peptides forming nanodiscs
S. R. Midtgaard, M. C. Pedersen et al. Soft Matter, 2014, 10, 738-752
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Ab-initio Modelling Modelling of particles with no prior information
Build complex models from uniform blocks
like LEGO but we use Dummy Atoms
Acts as a placeholder for, but does not resemble, a real atom
Occupies a known position in space
Has a known scattering pattern (Solvent or Particle)
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Ab-initio Modelling
Must be Compact
Must NOT be disconnected
Must NOT be Loose
Modelling of particles with no prior information
Build complex models from uniform blocks
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Ab-initio Modelling
With multiple data sets multi phase models can be produced
MONSA
Points in the search grid can be assigned to Different phases
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Ab-initio Example
SAXS data + crystal and
NMR structures show the N-
terminal domains of mouse
cMyBP-C form an extended
structure with a defined
disposition of the modules
However mixing with actin
results in a dramatic
increase in scattering signal
due to the formation of a
large, rod-shaped assembly
150
Å
Whitten, Jeffries et al. (2008) PNAS 105, 18360
Thanks to J Trewhella for these slides
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Ab-initio Example
Whitten, Jeffries et al. (2008) PNAS 105, 18360
Thanks to J Trewhella for these slides
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Ab-initio Example
Thanks to J Trewhella for these slides
Whitten, Jeffries et al. (2008) PNAS 105, 18360
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Ab-initio Example
Whitten, Jeffries et al. (2008) PNAS 105, 18360
Thanks to J Trewhella for these slides
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Idealised Solution SAS Experiment
Solution Scattering Data from Protein of Interest
Black Box
Neutron or X-ray source
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What exactly is the Black box?
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What exactly is the Black box?
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What exactly is the Black box?
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What exactly is the Black box?
Energy 7-15 keV, standard: 12.5 keV
q-range 0.020 - 5 nm-1
beam size at sample about 700 µm x 700 µm
flux at sample 3 x 1012 photons/s
detector distance 2.867 m
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Idealised Solution SAS Experiment
Solution Scattering Data from Protein of Interest
Black Box
Neutron or X-ray source
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Experimental Procedure
Clean Water Detergent Water Dry Load New Sample/Buffer Interlock Measure
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Manual sample handling
EMBL-HH 1st gen SC X33@DORIS
Evaluation Setup ID14-3@ESRF
2nd Gen SC ID14-3@ESRF
2nd Gen SC BM29@ESRF
2nd Gen SC BM29@ESRF P12@PETRAIII B21@Diamond
SC development
2007
2009
2010
2012
Sample Volume Cleaning time
Total cycle time
Reliability Confidence Throughput
Now
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Sample Preparation
In solution SAXS we observe the Average
Average
Monodisperse Mixture
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Region merged for background
Online SEC in ISPyB
Buffer
Subtracted
frames
Automatic Processing
Scattering returns to baseline
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Online SEC in ISPyB
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Current status of data collection:
Temperature
• Data acquisition between 4 and 60 C
• SEC operation at 4 or 20 degrees C
Exposure Time
• Standard 1 FPS (10 frames for Static)
• S200 column ~1 hour (3600 frames)
• Increase column ~10 mins (600 frames)
Sample Volume
• Minimum recommended 30 µL per measurement
• Approx. 5 mg/mL
• 100 µL stock recommended
• for static and SEC
Automated valve To switch between
SEC and Static modes
Gives users control
Safe and reliable switching
Maximises efficiency
cleaning between SEC runs
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SC compatible Microfluidics: First results Proof of Principle achieved
200 µm Background improved with scatterless pinhole
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SC compatible Microfluidics
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Experimental preparation and efficient use of beamtime facilitated through ISPyB
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Improved feedback for experimental preparation
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Improved feedback for experimental preparation
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Improved feedback with ISPyB
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Improved feedback with ISPyB
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Improved feedback with ISPyB
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Improved feedback with ISPyB
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Idealised Solution SAS Experiment
Solution Scattering Data from Protein of Interest
Black Box
Neutron or X-ray source
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Automated data acquisition
With feedback
The black box should not be scary
It should be reasuring that it is accessible
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Acknowledgments
GRENOBLE