Small-angle X-ray scattering
Get the overall size and shape of molecules in solution.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1Scattering from molecules in solution
When an X-ray beam passes through a protein solution, every electron scatters a little. Because the molecules tumble freely, the pattern on the detector is not a set of spots but a smooth, circularly symmetric glow that is brightest at the smallest angles and fades outward.
2What the curve tells you
- Guinier region (low q)
- For qRg below about 1.3 for a compact particle, ln I falls linearly with q². The slope gives the radius of gyration Rg; the intercept gives I(0).
- I(0) and mass
- On an absolute scale, I(0) divided by concentration is proportional to molecular mass, so SAXS reports whether the protein is a monomer, a dimer or a mixture.
- Kratky plot (middle q)
- q²I against q: a compact folded protein gives a bell-shaped peak that returns towards zero; an unfolded or flexible one rises to a plateau.
- P(r) (all q)
- The distribution of distances within the particle, obtained by an indirect Fourier transform; it falls to zero at the maximum dimension, Dmax.
3How it fails
Because SAXS sees everything in the beam and weights each particle by the square of its mass, small amounts of large particles dominate the low-angle data. A curve can also be distorted by the particles interacting with each other at high concentration, by the X-rays themselves damaging the sample, or by a buffer blank that is not exactly the sample's own.
Key terms
Practise these as flashcards →Scattering vector, 4π sin θ / λ; small q corresponds to large distances in the particle.
Root-mean-square distance of the particle's scattering mass from its centre.
The largest distance within the particle, where P(r) falls to zero.
The modulation of the scattering by interactions between particles; equal to 1 in an ideal dilute solution.
Removing the scattering of solvent and cell by measuring the exact matching buffer under identical conditions.
Measuring SAXS continuously as the sample elutes from a size-exclusion column, so each peak is measured free of aggregates.
When to use it
- Checking that a protein is a monodisperse monomer (or the expected oligomer) in solution before crystallisation or cryo-EM.
- Testing whether a crystal structure, a model or a complex matches the shape in solution.
- Following conformational change on ligand binding, or characterising flexible and intrinsically disordered proteins that no high-resolution method can capture.
Compared with alternatives
A quick check of hydrodynamic size and aggregation with a benchtop instrument, but no shape information.
Accurate mass and oligomeric state across an elution profile; complementary to the shape from SAXS.
Atomic structure, but of the molecule as packed in a crystal; SAXS checks whether that conformation holds in solution.
High-resolution 3D structure of large complexes; SAXS is faster and needs no grids, but gives only the overall envelope.