Small Angle X-ray Scattering

Why do you want to SAXS?

A practical introduction
 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
Small Angle X-ray Scattering

•  Crystallography supplies structural information

•  Describes low energy conformations of the

macromolecules within the crystal lattices

•  Critical for mechanistic analyses

SAXS offers complementary information

 SAXS
Small Angle X-ray Scattering
•  Information about:
 Macromolecular folding
 Unfolding, aggregation
 Extended conformations
 Flexible linked domains
In solution
 Shape, conformation
 Assembly state
 SAXS
Small Angle X-ray Scattering
•  Information about:
 Macromolecular folding
 Unfolding, aggregation
 Lowerconformations
 Extended resolution range of about
50Å to 10Å
 Flexible linked domains
In
resolution solution

 Shape, conformation
 No size limitations (NMR and EM)
 Assembly state
 SAXS
Small Angle X-ray Scattering

•  Information:
 Modelling allosteric mechanisms
 Supramolecular complexes
 Dynamic molecular machines

Processes ranging from eukaryotic DNA replication,

recombination and repair to microbial membrane
secretion and assembly systems
 SAXS
Small Angle X-ray Scattering

Flexibility and conformational changes

have become increasingly relevant for
accurate understanding, simulation, and
prediction of mechanism in structural
cell biology and nanotechnology
 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: the basics  
Elastic scattering

Wave interference

SAXS                                                                                                                                                                                                                                                      X-­‐ray  
                                                         Crystallography  
 SAXS: the basics

Data radially symmetric

(Isotropic)
Randomly oriented
distribution of
particles in solution

Buffer Sample

 SAXS is a contrast method where the scattering

signal is derived from the difference in the average
electron density, ρr(r), of the protein molecules of
interest, ρ(r), and the bulk solvent ρs (buffer)









Δρ  (r)=  ρ(r)-­‐ρs  
 SAXS: the basics

Buffer Sample

 The scattering curve, I(q), comes from the subtraction

of the buffer from the sample

  I(q) is a function of the momentum

Data  points  
transfer*
q=(2π sinθ)/λ (Å-1, nm-1)

*the directional momentum change that the photons undergo

 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: Sample preparation"

(ESSENTIAL)
•  Macromolecule sample and exact buffer
•  The sample cell takes 15 ul. 20 ul is safer
when using the robot
•  Minimum concentration is 1 mg/ml
•  Maximal concentration is 10 mg/ml
•  Identical buffer required
 SAXS: Sample preparation

 The determination of a macromolecular

SAXS profile requires at least two
experimental measurements:

1.  One measurement for the "buffer blank”: the

sample containing only the solvent without
the macromolecule

2. The other measurement for the sample

containing both the macromolecule and
solvent
 SAXS: Sample preparation
The Buffer
  Do not use detergents in the entire protein preparation
  Only a few cases (~1%, bellow CMC concentration) where the detergent
did not affect the protein signal

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

  The most common problem is aggregation in the sample

  Larger particles scatter X-rays more strongly than small

particles

  Aggregation will bias the results

  Strongly recommend doing either DLS, native gel, or gel

filtration (best)

  If your sample has a tendency to aggregate over time

àprepare the sample at diluted concentrations and then
concentrate it just before data collection
 SAXS: Sample preparation
CONCENTRATION
  The higher the concentration, the better the signal
  A balance between problems with aggregation/
oligomerization at the higher concentrations, unless the
macromolecule is well-behaved
  1-5 mg/ml and doing a concentration series
  Ideally data should be collected on at least three different
concentration to identify any concentration dependent
behavior
  Aggregation precludes data analyses
  Overestimation is a common problem
SAMPLE HANDLING
  96 well plate sample format @ SSRL beamline. Operates
with pipetting robot
 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: the beam line (4-2, SSRL)
 SAXS: the experiment
RADIATION DAMAGE
 This happens, maybe 1 in 10 samples
 Changes in the SAXS scattering curve due
to radiation damage
 Glycerol is a pretty good radical
scavenger, so 5% glycerol can work
TEMPERATURE
 The beamline was equipped with a Peltier
that can vary the temperature from 0 to
50ºC
 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: the data  

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

  Combined during data   A single SAX scattering

reduction to produce a single curve that is
SAX scattering curve that is
representative of the entire representative of the entire
sample sample
 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
Pos / Cons
  A single SAX scattering
  Combined during data
To merge or notcurve
o merge…
reduction to produce a single
that is
SAX scattering curve that is
representative of the entire
representative of the entire
sample 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 analysis. The Guinier Plot
  The lowest resolution portion of the SAXS data curve is
dictated by a single size parameter, the radius of gyration
(RG, the square root of the average distance of each scatterer
from the particle center)
  The Guinier plot of log(I(q)) against q2 will give a straight line
from which RG and I(0) can be extracted
 SAXS: data analysis. The Guinier Plot

Lack of linearity is a sign:

1. More care with the data
evaluation Repulsion  
2. Sample is elongated
3. Should not vary with
concentration
4. Interparticle interference
5. Aggregation
6. Radiation damage
 SAXS: data analysis. The Guinier Plot
@ Low q values
  I(0), intensity measured a zero angle (q=0)
  It is determined by extrapolation (coincident with the direct beam)
  I(0) is the square of the number of electrons in the scatterer 
sensitive to the assembly state
  It is unaffected by particle shape
  Useful for Mw determination

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

 Better estimation for samples complicated

by small amounts of aggregation

 Initial estimation of the shape

 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: Ab-initio SAXS envelope
 Dummy atoms/beads model to explain the
experimental data/scattering curves
 The programs try to minimize the discrepancy
function by applying the non-compactness
penalty using SA starting by random
configuration
 Optional use of symmetry constrains
 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 to aid Crystallization
 Sample quality (sample folding, assembly
and aggregation)
 SAXS curves to distinguish between samples
where aggregation can be ameliorated by
varying conditions, centrifugation, GF, from
the ones that are hopelessly aggregated
 Control of the molecular assembly state
 Identification of buffer conditions that help to
stabilize particular assemblies
 SAXS to aid Crystallization
 SAXS is sensitive to the overall shape of the
macromolecule  Kratky plot
 Important for engineering and characterizing
truncation mutations α-subunit of DNA
polymerase III (Lamers et al, 2006)
 Characterization of natively unfolded proteins
(Shell et al, 2007)
 SAXS
Small Angle X-ray Scattering

1. The sample preparation

2. The experiment

3. The data

4. Data reduction

5. Data analysis

6. Modelling

7. SAXS and crystallization

8. SAXS and crystallography
 SAXS to aid Crystallography
 Low-resolution SAXS envelopes for MR
phasing
FSEARCH
 AMoRe

  ASAXS  Advantage of the anomalous

signal  Heavy atoms location  Phasing
 SAXS to aid Crystallography

 Identification of biological interactions

Different  interacBons  
Summary
My SAXS data