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• Information:
Modelling allosteric mechanisms
Supramolecular complexes
Dynamic molecular machines
SAXS
X-‐ray
Crystallography
SAXS: the basics
Buffer Sample
Buffer Sample
DIALYSIS
Strongly recommend dialyzing sample
The difference between the scatter of the macromolecule and buffer is so
low, that simply making up the "equivalent" buffer is not sufficient to get
accurate subtraction (Hampton dialysis buttons, 30-50 ul size)
Salt increases the background. Concentration of the macromolecule has
more of an impact on signal than the buffer, so if the sample is
monodisperse in high salt, put it in high salt (up to 1M)
SAXS: Sample preparation
MONODISPERSITY
Buffer Sample
SAXS
Small Angle X-ray Scattering
1. The basics
2. The sample preparation
3. The experiment
4. The data
5. Data reduction
6. Data analysis
7. Modelling
8. SAXS and crystallization
9. SAXS and crystallography
SAXS: data reduction
http://
www.embl-
hamburg.de/
biosaxs/
software.html
SAXS: data reduction
h7p://situs.biomachina.org/
SAXS: data reduction
Two different exposures of
the sample in order to A single exposure of the
accurately measure both the
ultra-low and moderate sample
angle X-ray scattering of the
macromolecular sample
Oligomerization State
@ High q values
Details of the molecular shape
SAXS: data analysis. The Kratky Plot
• Identification of unfolded samples
• Globular macromolecules have bell-shaped
curves (parabola)
@ High q values
I(q) α q-4
Atomic resolution
information begins to
contribute
SAXS: data analysis
Pair-Distribution Function P(r)
Directly calculated through a Fourier transform of the
scattering curve I(q) into real space
Provides direct information about the distances between
electrons from the scattering sample similar to the
Patterson function (frecuency of vector lenghts)
P(r) is radially averaged and lacks the vectors corresponding
to vectors between scattering particles origin peaks and
pure translations in the Patterson function
SAXS: data analysis
Pair-Distribution Function P(r)
P(r)=0 @ r=0, r≥Dmax
Dmax, the maximum linear
dimension
Indication of the data quality
Unfolded proteins are often not zero at r=0
Sometimes difficult to determine Dmax
(extended structures, globular structures with
disordered extensions)
Aggregation or wrong background
substration
SAXS: data analysis
Pair-Distribution Function P(r)
Calculation of the Rg and I(0) using all the
collected data
Different
interacBons
Summary
My SAXS data