An Overview of Biological SAXS

An Overview of Biological SAXS:
Instrumentation,Techniques and Applications
June 14, 2012
Welcome
Peter Laggner
Director
Nanostructure Solutions
Bruker AXS - Austria
peter.laggner@bruker.at
+43 664 3002738
Brian Jones
Sr. Applications Scientist, XRD
Bruker AXS Inc. – Wisconsin, USA
brian.jones@bruker-axs.com
+1.608.276.3088
2
Pioneers of (Bio-)SAXS
Otto Kratky developed the
first commercially successful
SAXS camera
Andre Guinier – Concept of
particle scattering – radius of
gyration
Andre Guinier
Otto Kratky
3
Contents
•
What is SAXS?
•
What can SAXS do for biology?
• Biopolymer structure – proteins and beyond
• Membrane structure and dynamics
•
The ‚Hybrid Analysis Approach‘
• SAXS and Crystallography
• SAXS and Calorimetry
• SAXS and NMR
• SAXS and EM
• Lab and Synchrotron SAXS
•
What is needed in instrumentation?
• Optics: flux and resolution, background optimization
• Sample environment
• Software
•
Summary of the Bruker SAXS product range
4
SAXS - X-Ray Scattering in Transmission
> 6°
XRD/WAXS
X-Ray Beam
Internal structure,
Crystal parameters
0.02 - 6°
SAXS
Particle size/shape
Nanosized pores,
Defects
5
The Length Scales
x 106
nm
SWAXS
Liposomal
Carrier
This is where all the
chemistry happens
µm
Porous mineral
mm
The
Product
This is our reality
6
Typical Time Scales
-6
-4
-2
Synchrotron beamlines
0
2
log t (s)
Lab instruments
7
The Reciprocity: Size – Scattering Angle
Small
particles
Large
particles
Wide SAXS range
Narrow SAXS range
8
Why is SAXS Special?
The angular resolution needs to be much better for SAXS than for XRD –
•
only ~ 6° ROI for two decades in real space
50
WAXS
40
angle (degrees)
•
30
Molecular Lattice
Unit cell dimensions
symmetry
20
Nano-Envelope
Size / Shape
10
SAXS
0
10
100
1000
distance (A)
9
Advantages of Simultaneous
SAXS and WAXS
crystalline
WAXS
SAXS
Scattering
curve
amorphous
150
nm
Nanoscale
domains/particles/
pores
1
10
Angström
3
Atomic / molecular scale
10
Where Can SAXS Help in Biology?
• Drug Discovery – Proteomics – Structural Biology
• Drug Development – Formulation – Process Control
• Biomaterials - Hair, Skin, Bone, Tissue Engineering
• Biochem / Biophys Basic Research
11
Biochem/Biophys Basic Research
The most important applications of SAXS in basic research relate to:
•
Macro- / supramolecular interactions in solution with small
molecules, salts , (Hofmeister series …)
•
Supramolecular complex formation in solution (protein-lipid, proteinnucleic acid,…)
•
Self-assembly of amphiphilic compounds (Micelle formation ,
coagulation…)
Laboratory SAXS/SWAXS/GISAXS become
complementary standards to spectroscopic and
thermodynamic tools in biophysics/biochemistry.
Powerful instruments provide the necessary speed
of measurement.
12
Proteomics – Structural Biology
Q: Drug binding effect on enzyme structure in solution?
 SAXS in dilute solution – radius of gyration (RG) , molecular weight , max.
dimension
Q: Search for optimum crystallization conditions?
 SAXS in different salt or polymer solutions – size (RG) monitors
attractive/repulsive interactions
Q: Differences between X-ray crystal structure and solution structure?
 SAXS curve fitting with crystal date (e.g. from PDB data bank)
Q: Oligomerization / multi-protein assembly in solution?
 SAXS under different protein concentrations – how to bring more protein into
1 ml ?
13
SAXS in Structural Proteomics and
Drug Discovery
Genomics
Protein
Synthesis
Candidate
Selection
Protein
Structure
Rational
drug design
Protein sizing to determine:
• State of aggregation in solution
(monodisperse/polydisperse)?
• Suitability for crystallization?
• Effects of salts, additives, ligands?
14
Biomaterials: Hair, Skin, Bone, Wood…
NANOGRAPHY :
This is the domain of NANOSTAR
Small- and wide-angle patterns of oriented
samples – 2D detection / sample scanning
15
BioSAXS Sample - Practice
Samples typically consist of biological macromolecules and
their complexes (proteins, DNA and RNA) in solution

Homogeneous

Monodisperse

Concentration: >1 mg/ml

Sample amount: 10-15 ml

Perfectly matched buffer solution provided for solvent
blank measurement
16
SAXS Information on Protein Structure
experimental data
theoretical profile from atomic model
shape
log I
?
folding
atomic structure
50 Å
0,0
0,1
0,2
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
0,3
-1
q[Å ]
0,4
13 Å
17
Particle Size – Guinier Law
Guinier law:
2 2
𝐼(q) = I(0).𝑒 −𝑅 q /3
𝑙𝑛 𝐼 𝑞 = 𝑙𝑛𝐼(0) − 𝑅2 q2/3
Valid up to q.R ≤ 1,2
•
•
•
Guinier plot:
Rg, radius of gyration.
I(0), Molecular weight
Slope = - ln(10) . R2/3
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
18
Protein Folding - Kratky Plot
The appearance of the
SAXS curve in the
Kratky plot allows to
infer chain folding
characteristics -
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
19
Fourier Transformation – The P(r) Function
1
sin( qr )
P( r ) 
dq
 I ( q).q
2
qr

2
2
0
Limit:
qmin.Dmax ≤ 𝜋
𝑃 𝑟 = 𝑇( 𝐼(𝑞))
The Pair Distance Distribution Function P(r) is the Fourier Transform of the SAXS
curve – it is a real-space function and hence intuitively better suited for modelling
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
20
Pair Distance Distribution Function P(r)
Maximum particle dimension, general shape
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
21
SAXS Size Range
Limit:
qmin.Dmax ≤
𝜋
Putnam and Hammel et
al. 2007 Q R Biophysics
40:191-285
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
22
Solution Structure Modeling
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
23
Proteomic Scale, Shape and Assembly
Hura, Menon, Hammel et al.
(2009) Nature Methods 6,
606 - 61
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
24
In 1971: 10 min/point
25
Today:
Protein Size and Shape Within Minutes by Lab-SAXS
3 min
Pair Distance
Distribution
12 min
6 min
15 min
9 min
18 min
(Bruker MICROPix™)
Stable results after < 12 min exposure
Size (Radius of Gyration) to < +/- 3 % within 3 min
Sample volume < 10 µl; concentration 0.1% -
26
Speed of SAXS
Dilute protein solution SAXS in human times – one coffee break
0.4%, Exposure 30 min
ALD
BSA
THI
Lyso
qmin = 0.016 Å1
d = 400 Å
27
Speed of Analysis – BSA model in 15 min
Experimental vs
fitted I(q)
Crystal Structure
SAXS
p(R)
DIMER
28
Conclusions
•
Protein SAXS does not require synchrotron radiation
•
Valuable SAXS data can be obtained in 30 min or less
•
Radii of Gyration can be determined in less than 5 min
•
Data of sufficient quality for informative molecular modeling
can be obtained in the home laboratory
29
Lipidic Formulations
Membrane Biophysics
Q: Polymorphic structure and transitions of liposomal formulations?
 Simultaneous small-and wide-angle scattering (SWAXS) in
T-/c-/p- scanning mode
Q: Interactions between lipid model systems and drugs?
 Dose-dependent SWAXS scanning
Q: Simultaneous calorimetric and structural measurement?
 Integrated SWAXS – scanning microcalorimetry
Q: Solid-supported thin lipid films? ORIENTED! 2D-pattern
 Grazing-incidence SAXS (GISAXS)
30
Liposomes and Lipoplexes
a Rich Field for SAXS/WAXS
Starting from the
components:
Lipid self-assembly
31
Thermal Phase Transitions
SWAXS T-Scans
0.5°/min, 1 frame/min
Dipalmitoyl-PC (DPPC)
Phases: L‘ , P‘ , L
SAXS: lamellar stacking structure
WAXS: hydrocarbon
chain packing
5.0
48.0
50.0
53.0
54.0
61.3 56.0
53.0
56.0
5.0
0.00
0.01
0.02
0.03
0.04
0.05
0.06
s [1/Å]
32
Drug Liposome Interaction
DSC-trace
SAXS
33
Searching Targets for Anti-Viral Therapy
Q: Which parts of the protein are responsible for fusion?
How can viral fusion with the cell membrane be suppressed?
Viral Fusion Protein: MembranePeptide Screening
Viral entry:
Membrane
Fusion
Viral Fusion
Protein
P. Laggner, Ana J. Perez Berna and Jose Villalain, 2007
34
Host Membrane
Viral Membrane
260-277
197-214
274-298
309-326
435-452
400-417
351-368
25°
708-725
603-620
547-564
25°
0.1
0.1
0.2
0.3
0.4
0.1
0.2
0.3
0.4
0.1
0.2
0.3
0.4
0.1
0.2
0.3
0.4
0.1
0.2
0.3
0.2
0.3
0.4
0.5
0.1
0.2
-1
-1
q-axis (nm )
-1
q-axis (nm )
0.4
0.5
0.1
0.2
-1
q-axis (nm )
-1
q-axis (nm )
0.3
0.3
0.4
0.5
0.1
0.2
0.3
0.4
0.5
0.1
0.2
0.3
0.4
0.5
0.4
0.5
0.4
0.5
0.4
-1
-1
q-axis (nm )
q-axis (nm )
-1
q-axis (nm )
-1
q-axis (nm )
-1
q-axis (nm )
q-axis (nm )
45°
45°
0.1
0.2
0.3
0.4
0.1
0.2
-1
0.3
0.4
0.1
0.2
0.3
0.4
0.1
0.2
0.3
0.4
0.1
0.2
0.3
0.1
0.4
0.2
0.3
0.4
0.5
0.1
0.2
-1
q-axis (nm )
-1
-1
q-axis (nm )
0.3
0.4
0.5
0.1
0.2
-1
q-axis (nm )
-1
q-axis (nm )
0.3
0.4
0.5
0.1
0.2
-1
q-axis (nm )
0.3
0.4
0.5
0.1
0.2
-1
q-axis (nm )
0.3
-1
q-axis (nm )
q-axis (nm )
-1
q-axis (nm )
q-axis (nm )
70°
70°
0.1
0.2
0.3
0.4
0.1
0.2
-1
0.3
0.4
0.1
0.2
0.3
0.4
0.1
0.2
-1
0.3
0.1
0.4
0.2
50
Temperature (ºC)
60
70
10
30
40
0.4
0.5
0.1
0.2
50
Temperature (ºC)
60
70
10
30
40
0.4
0.5
0.1
0.2
-1
0.3
0.4
0.5
50
Temperature (ºC)
60
70
10
20
30
40
50
60
70
10
F10
F5
E24
Temperature (ºC)
0.1
0.2
-1
0.3
0.4
0.5
0.1
0.2
-1
q-axis (nm )
0.3
-1
q-axis (nm )
q-axis (nm )
Fusion Helper
E19
20
0.3
q-axis (nm )
q-axis (nm )
PreTM
E13
20
0.3
-1
q-axis (nm )
FP
40
0.2
-1
E11
E2
30
0.1
q-axis (nm )
-1
20
0.4
q-axis (nm )
q-axis (nm )
10
0.3
-1
q-axis (nm )
20
30
40
50
Temperature (ºC)
60
70
10
20
30
40
50
60
70
10
F27
20
30
40
50
60
70
10
F35
20
30
40
50
60
70
10
20
F49
30
40
50
60
70
10
20
30
40
50
60
70
Searching Targets for Anti-Viral Therapy
SAXS and DSC are suitable techniques for finding regions
that promote non-lamellar phases likely to be involved in
fusion.
Fusion Peptide
PreTM
The region 603-620, fusion helper, 274-298, fusion peptide,
and 309-326, PreTransmembrane domain are entry targets
against HCV (hepatitis C virus).
36
‚Hybrid Analysis‘
Getting the Bigger Picture
‚Hybrid Analysis‘ strategy combines methods and
techniques of
• Diffraction
• Spectroscopy
• Imaging
• Thermodynamics
to get the complete picture of the complex
structures, interactions and dynamics of biological
systems at the submicroscopic scale
37
‚Hybrid Analysis‘
Combining Methods
Diffraction
SAXS
DSC/SAXS
structure and
dynamics
SC-XRD
atomic resolution structure
Membrane receptor
Cryo-EM
large-scale shape
Spectroscopy
NMR
atomic resolution
structure in solution
tubulin
AFM
structure at surfaces
Imaging
MICRO-CT
leads in all these fields – except for EM
38
The Need for Lab-SAXS:
Just-In-Time Results
•
There is an increasing need for robust, simple, and powerful SAXS
instrumentation for the laboratory, because…
•
Bio- and Nanomaterials are precious – speed matters
•
SAXS can give unique information – noninvasively
•
Synchrotrons are not instantly available
and
• The Hybrid Analysis Approach cannot work
unless different methods are available
simultaneously around a given project
39
SAXS – Cryo-EM : 1012 vs 1 Molecule
SAXS on soluble antigen antobody
complex allowed to describe the average
arrangement of the Fab arms under
antigen binding - simply by analysing
the radius of gyration
Three different IgG molecules (A, B, C) modeled with
CryoET. Each model is shown in three different orientations.
From a collection of such models it was possible to derive a
potential energy model to describe the distribution of
angles observed among the Fab arms and Fc stem.
Courtesy of Bongini et al Proceedings of the National
Academy of Sciences 101:6466-6471; ©2004 PNAS
40
SAXS and NMR
Enhanced Accuracy
NMR structure of the tetraloop receptor complex
•
•
•
•
NOE distance restraints: 700 x 2
Intermolecular NOEs: 36 x 2
Residual dipolar couplings (RDCs): 11 x 2
RMSD 1.0 Å
• Davis et al., 2005. RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloop-receptor
complex. J. Mol. Biol. 351(2):371-82
• Davis et al., 2007. Role of metal ions in the tetraloop-receptor complex as analyzed by NMR.
J. Mol. Biol. 351(2):371-82
Courtesy of Sam Butcher‘s Lab, Department of Biochemistry, University of Wisconsin
41
SAXS and NMR
Can SAXS data substitute for intermolecular NOE and H-bond restraints?
Courtesy of Sam Butcher‘s Lab, Department of Biochemistry, University of Wisconsin
42
SAXS and NMR
RG
------------------------
•
•
•
NMR
NMR+SAXS
Measured
25.1
23.1
23.0
Courtesy of Sam Butcher‘s Lab, Department of Biochemistry, University of Wisconsin
43
SAXS and NMR
Conclusion / Hypothesis:
SAXS data can be used to define molecular
interfaces and can compensate for sparse NMR
data
Combination of SAXS + NMR data
should lead to even more accurate structures
44
Thin Film Structure by GISAXS
XRR
Reflection
and
refraction
Diffraction
detector
from multilayer optics
collimator
.
sample rotation axis
divergence < 1 mrad (0.057 °)
45
Lab-GISAXS of Phospholipid Mesophase
z
y
DOPC on Si chip
Hecus S3-MICROpix , 1000 s
This structure has highly promising features for
thin-film biotechnology:
• controlled pore size,
• large inner surface
• easy to produce
Can we functionalize it (e.g. cytotoxic)?
… tests with melittin (bee venom peptide)
46
Functionalized Peptide – Lipid Film
DOPC/Melittin R = 10-4 in vacuo on Si
20 hours later, air, RT
S3-MICROpix 10 min
Sqashed liposomes (4,6 nm)
Surface-aligned multilayer (4.8 nm)
• The original cubic film structure of DOPC is transformed into
lamellar, isotropic multilayers already at molar ratios of 1:10.000 of
melittin.
• Two (or more) domains with different lamellar repeats appear
47
The Synchrotron Revolution
SAXS Development over last 40 years
•
Bio-SAXS, especially protein SAXS has
tremendously grown with the
availability of SAXS beamlines
•
A major part of the synchrotron BioSAXS projects could be done equally
well at home-lab instruments, if
available
•
Synchrotron SAXS projects can be
much more productive if well
prepared by lab-SAXS
Synchrotron
Home-lab
1970
1990
2010
But‚ I am tired of travelling thousands of miles to a synchrotron to
get the information I need to do today, not in three or six months (a
biochemist and convert synchrotron user)
48
The MICRO™ Revolution
49
The MICRO™ - Platform
SAXS system for instant laboratory nanoanalytics
MICRO - SWAXS
MICRO - SAXS
Speed:
3 x higher flux than other lab-SAXS system by IµS-microsource
(INCOATEC) and MICRO™ Kratky-optics*
True SAXS :
No information loss through point-beam focusing / no de-smearing
Interactive :
Study ligand binding, salt effects, conformational changes on proteins
in the home laboratory, in situ, in real time
* Combination of HECUS Kratky system design with INCOATEC source / focussing optics and Bruker detectors
50
Kratky vs Pin-Hole Collimation
MICRO vs Nanostar
Defining slits
~500 mm
Cleaning slit
Pin-hole : 360° field of view
Kratky block: 180° field of view,
no background in measuring field
~60 mm
~300 mm
51
Detectors
• 1D SAXS and/or WAXS: VANTEC-1
• 2D SAXS/GISAXS: Dectris‘ Pilatus 100K
53
MICROpix – Sample Cells
• Liquid sample microcell for ambient
or non-ambient
• Microcell for pastes and powders
• Capillary-flow cell for liquids and
gases
54
Synchrotron-Proven Hard-and Software
•
Detectors:
Pilatus (Dectris 100K) – radiation hard, can stand primary beam
VANTEC-1 – synchrotron proven, fast 1-D detector
Serial scans – no image plate change-over
•
Software:
ATSAS suite: designed by most experienced protein SAXS expert
(D. Svergun, EMBL, Hamburg)
55
S3-MICROcaliX
Combining SWAX
&
Calorimetry*
• Calorimetry provides only the enthalpy e.g. of phase changes, no
structure information
• SAXS is essential to identify long-period structures, e.g. in fats
• Detection of pre-transitional changes in nanostructure – domains,
interfaces
• Simultaneous measurement is crucial with unstable materials
*Cooperation started with Michel Ollivon, († 2006) Greard Keller, Claudie Bourgeaux et al in 2000, continued with
Setaram, Caluire, France
56
WAXS/WAXS/DSC
The MICROcaliX™
Microcalorimeter
S3-MICROcaliX
Microbeam X-Ray Camera
Integrated Laboratory Benchtop
System
Developed in cooperaqtion with SETARAM Caluire, France
60‘‘ time-frames
57
Micro-Calorimeter Specifications
•
•
•
•
•
•
•
•
•
Sample volume: 20µl (open capillary)
Temperature range: -25 °C 200°C
(-30°C  200°C reached when RT~20°C)
Average sensitivity: 410µV/mW
Isothermal detection limit:1.5µW
Scanning detection limit: 2.5µW
Static drift (25 min isotherm at 26°C): 3µW
Repeatability< 1%
Maximum heating rate : 5K/min
Maximum cooling rate
 5K/min(200°C  50°C)
 1K/min (50°C  0°C)
 0.5K/min (0°C  -30°C)
MICROcaliX™ matches
the best standalone instruments
58
Speed of SAXS
Calorimetric SAXS : MICROcaliX™
Ag-behenate powder:
20°-200°C (1.5°/min)
1D-SAXS: 1 frame/min
2 D-SAXS pattern of Ag-behenate
heating scan: 110°C - 200°C
(1°C/min)
60 frames (1 min each, 1°C/frame).
Animation is not time-synchronous
with 1D (left)
59
Silver Behenate SAXS-DSC
with Bruker MicroCaliX
ICTP School Trieste 210312
60
Model of the ribbon phase
Two-dimensional centrered rectangular lattice with
space group cmm
b
a
61
Cocoa Butter: SAXS shows–pre-transitional nanodomain effects
Cocoa Butter Polymorphism
SWAXS/DSC with MICROcaliX™
SAXS
6.4 nm
4.4. nm: second phase
n=2
38°C
n=4
n=5
SAXS zoom
-10°C
Onset of transition
already at much
lower temperatures
than indicated by
calorimetry and by
WAXS
WAXS
simultaneous SAXS and WAXS
• scan 1°C/min
• signal strength sufficient to measure SWAXS with time resolution of ~ 20 s, i.e. faster scanning is technically
possible.
62
MICROcaliX™ – Fields of Application
Component
Food
Pharmaceutical
Bio
Others
Application
protein
denaturation, aggregation
Protein powder
crystallization
starch
gelatinisation, retrogradation, glass
transition
milk
Melting, crystallization, denaturation,
aggregation
fat, chocolate
Melting, crystallization, lipidic transition,
polymorphism
fat
hydrocolloids
crystallization
melting, gelation
sugar
Melting, crystallization, glass transition
(amorphism)
drug, excipient
Melting, polymorphism
protein
denaturation, aggregation, polymorphism
Lipid, membrane, vesicle
Lipidic transition
Liquid crystal
transitions
Gas hydrate
Formation, dissociation
63
Innovation with Integrity
© Copyright Bruker Corporation. All rights reserved.
64