Small Angle X-ray Scattering (SAXS): Nanoparticles can be characterized by SAXS. Applicable in biophysics. Protein folding

1. Small Angle X-ray Scattering
Kalyan Jyoti Kalita
16IP011
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CH-3206: Instrumentation in Chemistry

2. “When the scientist have learned how to control
the arrangement of matter at a very small
scale they will see materials take an
enormously richer variety of properties”
- Richard Feynmenn (1929)
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3. Overview
• Introduction
• History
• Applications of SAXS
• Structural information obtained by SAXS
(Guinier Plot)
• Instrumentation
• Advantage/Disadvantage
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4. • SAXS is an instrument to be capable of
delivering structural information of
macromolecules between 5 and 25 nm
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5. History
• Krisnamurty (1930): P. Krisnamurty, Ind. J.
Phys., 5, 473
• Hendricks(1932), Mark (1932), Warren (1936)
• A. Guinier(1937): Interpretation of
inhomogenities in Al alloys
• G. Porod(1942): Description of dense system
of colloidal particles, micelles, and fibers.
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6. Applications
• Size of particulate systems viz. Colloids,
globular proteins etc.
• Inhomogeneous structure such as polymer
chain
• Distorted crystalline structure like crystal of
soft matter.
• Gives information about macromolecular
folding, unfolding, aggregation, different
conformations.
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7. • Supramolecular complexes
• Dynamic molecular machines
• Critical for mechanistic analysis of proteins
• Nanomaterials
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Lee B. et al. Small Angle X-ray Scattering for Nanoparticle Research Chem.Rev., 2016, 116 (18),11128-11180Lee B. et al. Small Angle X-ray Scattering for Nanoparticle Research Chem.Rev., 2016, 116 (18),11128-11180

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Nano structured
materials
Nano structured
materials
Multilayer Films,
Polycrystalline,
Nanocomposites,
Patterned structures,
Bulk structures,
Liquid crystals,
Biological samples,
Fractals, Gels etc.
Monochromatic
X-Rays
Scattered
X-Rays
Lee B. et al. Small Angle X-ray Scattering for Nanoparticle Research Chem.Rev., 2016, 116 (18),11128-11180Lee B. et al. Small Angle X-ray Scattering for Nanoparticle Research Chem.Rev., 2016, 116 (18),11128-11180

9. • Technique where elastic scattering of X-rays
(wavelength 0.1-0.2 nm) are used
• Capable of delivering structural information of
macromolecules between 5 and 25 nm (in
partially ordered system upto 150 nm)
• Ultra-SAXS can resolve even larger dimensions
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T: X-ray source
S: Sample
�: Scattering angle
D: Detector
Fig: Essential parts of SAXS system

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The scattering curve,
I(q), comes from the
subtractionof the
buffer from the
sample.
q=(4π sinθ)/λ (Å-1,
nm-1)

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Guinier Plot
J. Appl. Cryst. (2016). 49, 1412-1419
• The Guinier region in small-angle X-ray
scattering (SAXS) defines the radius of
gyration, Rg, and the forward scattering
intensity, I(0)
• Radius of gyration: distribution of the
components of an object around an
axis
• Deviations : parameter errors,
problematic low-resolution data, some
kinds of intermolecular interactions or
elongated scatters.

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Curtesy: Inés Muñoz, Spanish National Cancer Research Centre

14. Sample preparation
• Proper buffer solution should be used
• Minimum concentration is 1 mg/ml
• Maximal concentration is 10 mg/ml
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15. SAXS vs XRD
• SAXS and x-ray diffraction are fundamentally similar.
• Both methods make use intense beam of x-rays to obtain
structural information about the sample.
• Differences arise from making measurements of target
molecules in solution (SAXS) or embedded in a crystal
(diffraction).
• X-ray diffraction from a crystal yields much higher resolution
and a better signal-to-noise ratio.
• SAXS analysis can be applied to flexible proteins that don’t
easily crystallize.
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16. SAXS Vs XRD
• SAXS data collection is rapid (seconds).
• SAXS requires microliters of a ~1-20 mg ml-1
solutionof protein. Very economical.
• SAXS in combination with x-ray
crystallographic data can be very powerful for
the analysis of large multi component
systems.
• SAXS requires microliters of a ~1-20 mg ml-1
solution of protein. Very economical.
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17. • Alba K, Bingham RJ, Kontogiorgos V. Mesoscopic structure of
pectin in solution. Biopolymers. 2017;107:e23016.
Mesoscopic: materials of an intermediate length. The scale of
these materials can be described as being between the size of
an atom and of materials measuring micrometres. (Bruker
nanostar 1-125 nm)
Pectin is a structural heteropolysacharide contained in the
primary cell walls of terrestrial plants
Slide No17

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Alba K, Bingham RJ, Kontogiorgos V. Mesoscopic structure of pectin in solution. Biopolymers. 2017;107:e23016Alba K, Bingham RJ, Kontogiorgos V. Mesoscopic structure of pectin in solution. Biopolymers. 2017;107:e23016
a) Sample b) with NaCl

19. Advantage/Disadvantage
• Advantage of SAXS over crystallography is
that a crystalline sample is not needed.
• Particle range: Bruker NANOSTAR (1-125 nm)
• Kind of a complementary technique
• Cost of SAXS: High
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20. References
1. David A. Jacques and Jill Trewhella, Protein Science; 2010;
19; 642—657
2. Christopher D. Putnam et al. X-ray solution scattering (SAXS)
combined with crystallography and computation: defining
accurate macromolecular structures, conformations and
assemblies in solution, Quaterly Reviews in Biophysics, 2017
3. Christopher D. Putnam Guinier peak analysis for visual and
automated inspection of small-angle X-ray scattering data J.
Appl. Cryst. (2016). 49, 1412-1419
4. http://smallangle.org/
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