SAXS Analysis
in Structural Biology
How SAXS Benefits
Structural Biology
Small-Angle X-ray Scattering, SAXS for short,
is an increasingly popular technique for
structural investigations in molecular biology,
offering valuable complementary information
to established techniques.
Common methods for structure analysis on biological
macromolecules and complexes thereof are crystallography
and Nuclear Magnetic Resonance (NMR). SAXS complements
both these high-resolution techniques with valuable additional
information – and offers specific benefits compared to
crystallography.
Requiring a single crystal of a biomolecule, crystallography often
presents a challenge – and the required “frozen” (crystallized)
state of samples leaves essential questions unanswered: How
is the sample's structure influenced by its natural environment?
Which dynamic processes is the sample involved in?
NMR and SAXS answer these questions. While NMR delivers
high-resolution structure information, its signal is often so
complex that it cannot be interpreted without further input.
Employing SAXS, biological macromolecules can be investigated
in solution, under physiological conditions. Analyzing samples
The birthplace of SAXS analysis
in their native state is essential to studying dynamic processes
such as structural changes upon ligand binding, or protein
folding/unfolding upon environmental changes.
Based on its rich output of structural information,
with easy sample preparation and flexible experimental
conditions as additional benefits, SAXS is a key technique
1957
First commercial instrument for SAXS
analysis built by Prof. Otto Kratky
together with Anton Paar in Graz,
Austria
1981
Kratky compact camera
developed, over 800
instruments produced
by Anton Paar and sold
worldwide
for biological research.
SAXS: Basic principle
A SAXS measurement determines the scattering intensity
of a molecule as a function of the scattering angle, typically
at resolutions from 1 nm to 200 nm. When X-rays penetrate
a solid or liquid material containing nano-sized domains of
another they are scattered by the nanostructures – in small
angles, since these structures are large compared to the
X-rays' wavelength. The resulting scattering patterns are
characteristic for particles’ size, shape and internal structure.
Tissue transglutaminase
SAXS Results at a Glance
Size and shape of biological
macromolecules
3D envelopes of proteins or
protein complexes
Degree of aggregation
Mass analysis
Folding and unfolding of proteins
Protein stability upon variation
of external parameters
Structure changes due to
ligand binding
2003
Introduction of
SAXSess instrument
featuring X-ray beam
monochromatization
and 2D detection
2009
2012
Introduction of
SAXSess mc2,
offering a variety
of X-ray sources
and detectors
Scattered intensity I(q)
Shape
Overall fold
Atomic structure
Introduction of
SAXSpace, the
first self-aligning
SAXS analyzer
Scattering vector q [nm-1]
Monochromatic X-ray beam
Scatt
ered
Small amount
of protein sample
X-ray
s
Protein Analysis
SAXSpace
The SAXSpace nanostructure analyzer is tailored to
research on proteins, protein complexes, DNA and
other biological macromolecules.
Short measuring times:
high-throughput screening
Small sample volume requirement
Compact design for high intensity
and ease of use
SAXSpace covers all Bio-SAXS applications.
The powerful SAXSquant™ software provides you
with several analysis options for the measured
scattering data.
Determine protein size and mass
study geometrical characteristics of proteins by means
of the limit at which the scattering vector tends to zero.
The radius of gyration (proportional to the particle size) is
In (I(q))
The well-established Guinier extrapolation allows you to
small
large
determined from the initial slope of the scattering curve
in the Guinier plot. Based on this, the particle volume
(proportional to the particle mass) is then obtained from
the ordinate intercept.
q2[nm-2]
Monitor protein unfolding
unfolded
The limit of large scattering angles provides you with information about
I(q)*q2
protein folding and unfolding. The Kratky plot makes use of the fact that the
scattering curve of compact particles decays with the power of -4 at large
folded
angles, while random walk polymers only decay with the power of -2. In a plot
I(q) q2 vs. q, the signal of an unfolded protein runs into a plateau, while a folded
and compact protein shows a distinct maximum. In this way, time-resolved
q
SAXS experiments can be used to follow protein denaturation processes.
All Possibilities
Powerful software
The powerful SAXSdrive™ and SAXSquant™
software packages fully control all SAXSpace
system components and facilitate data acquisition,
processing and analysis.
Optimized for high-throughput screening
Simple data processing via fully
customizable templates
Covers all essential evaluation routines
Easy data transfer to ab-initio modeling
programs (e.g. ATSAS, SasView)
Create 3D envelopes of globular proteins
The Fourier transformation of SAXS curves uncovers information in real space
and gives you access to the Pair Distance Distribution Function.
The distribution of distances between scattering centers (electrons) within a
particle gives rise to interference phenomena which produce the actual scattering
pattern. The Pair Distance Distribution Function itself allows you to extract valuable
information about particles’ size, shape and internal structure. More sophisticated
ab-initio methods tailored to protein data even produce low-resolution envelopes
and bead models or show the folding of the protein backbone.
Model the structure of protein-protein /
protein-DNA complexes
Prior information obtained using other techniques (e.g. protein crystallography)
can be combined with SAXS data to determine the relative orientation and
placement of individual domains in a complex. This process is known as rigid
body modeling. This approach enables you to find structures of complexes that
best fit your experimental data. For example, the structure of a protein complex
can be solved with the aid of the constituting proteins’ crystal structure.
SAXSpace:
Your Benefits
High quality and efficiency
combined
SAXSpace employs an intense monochromatic X-ray
beam with extremely low background radiation,
providing reliable data even on the most low-contrast
proteins. This beam quality is achieved with an
advanced scatterless collimation system, based on
the Kratky block collimator which was developed by
SAXS “founding father” Prof. Otto Kratky together
with Anton Paar in the 1950s.
ASX-c autosampler
High throughput
The ASX-c autosampler enables high-throughput screening,
automatically measuring up to 192 samples and protecting
sensitive proteins with an integrated cooling option: Samples are
stored at low temperatures while waiting for the measurement.
Smallest sample volumes
Precious protein samples are in good hands with the µ-Cell:
It requires less than 10 µL of sample for a complete analysis
and offers full sample recovery.
Short measuring times
SAXSpace is the only SAXS system to optionally employ a linecollimated X-ray beam. This enables the illumination of a large
volume representative of the whole sample, so measuring times
are significantly reduced and local radiation damage is avoided.
Find more information on collimation types in the right.
Easy handling
SAXSpace particularly stands out for its ease of operation.
The system aligns itself automatically which allows users to
quickly and easily obtain SAXS results on protein samples.
Once installed, the compact system delivers reliable results, with
a high sample throughput and minimal maintenance required.
µ-Cell
The Right Collimation
for Each Application
Point collimation is the right choice for analyzing anisotropic samples
Point or line One system, two options
and solid samples with inhomogeneous structures. Line collimation
is advantageous for measuring liquids, especially when the
scattering signals are low, which is the case with protein solutions.
Both collimation types have distinct applicationspecific advantages: SAXSpace offers you both and
lets you decide.
The line-collimated incident X-ray beam hits a large sample volume
and illuminates a large number of proteins. This gives you a high
scattering signal while keeping the radiation exposure on individual
protein molecules low. In this way, line collimation significantly
In SAXS analysis, X-rays are transformed into a
well-defined beam – a point or a line – in a process
called collimation. Common SAXS systems employ
point collimation only.
reduces measuring times compared to point collimation.
For isotropic samples without orientation effects, results measured
using line collimation are subjected to a desmearing calculation,
to ensure that no information is lost. Various desmearing routines
Based on its advanced Kratky-based collimation
system, SAXSpace is is the only SAXS system that
enables you to switch quickly between point and
line collimation.
have been developed over the last few decades and have been
successfully applied in studies on a vast number of different
sample types, as can be found in literature. The example below
demonstrates the accuracy of this procedure.
SAXSpace and synchrotron
data compared
This figure compares data measured at a dedicated Bio-SAXS
10 0
synchrotron beam line (point collimation) and data obtained using
SAXSpace (line collimation). After desmearing the line-smeared data,
the two results overlap nicely across the entire measured q range.
After only 30 minutes of measuring time, SAXSpace generates the
I(q)normalized
10 -1
same data quality as the synchrotron instrument. Obviously, all
evaluation methods applicable to synchrotron data can also be
used for data obtained with SAXSpace.
10 -2
10 -3
Diluted protein solution
SAXSpace, 30 min, line collimation
SAXSpace, 30 min, corrected
Bio-SAXS beam line at EMBL Hamburg, 2 min
10 -4
0.1
1
q[nm-1]
10
Anton Paar® GmbH
Anton-Paar-Str. 20, A-8054 Graz
Austria - Europe
Tel.: +43 (0)316 257-0
Fax: +43 (0)316 257-257
E-mail: [email protected]
Web: www.anton-paar.com
Specifications subject to change without notice. | 05/13 D21IP007EN-A