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June 14, 2012
An Overview of Biological SAXS: Instrumentation,Techniques and Applications
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Peter Laggner Director Nanostructure Solutions Bruker AXS - Austria [email protected] +43 664 3002738
Welcome
Brian Jones Sr. Applications Scientist, XRD Bruker AXS Inc. – Wisconsin, USA [email protected] +1.608.276.3088
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Andre Guinier Otto Kratky
Pioneers of (Bio-)SAXS
Otto Kratky developed the first commercially successful SAXS camera Andre Guinier – Concept of particle scattering – radius of gyration
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• 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
Contents
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SAXS - X-Ray Scattering in Transmission
> 6°
SAXS
Particle size/shape
Nanosized pores,
Defects
XRD/WAXS Internal structure, Crystal parameters
0.02 - 6°
X-Ray Beam
6
nm µm mm
Porous mineral
This is where all the chemistry happens
Liposomal Carrier
The Product
This is our reality
x 106
SWAXS
The Length Scales
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Typical Time Scales
log t (s) -6 -4 -2 0 2
Synchrotron beamlines Lab instruments
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The Reciprocity: Size – Scattering Angle
Wide SAXS range
Narrow SAXS range Large particles
Small particles
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Why is SAXS Special?
10 100 1000
0
10
20
30
40
50
an
gle
(de
gre
es)
distance (A)
Nano-Envelope
Size / Shape
SAXS
WAXS
Molecular Lattice
Unit cell dimensions
symmetry
• The angular resolution needs to be much better for SAXS than for XRD –
• only ~ 6° ROI for two decades in real space
10
SAXS
1 150 nm 10 3 Angström
WAXS crystalline
amorphous
Scattering curve
Nanoscale domains/particles/
pores
Atomic / molecular scale
Advantages of Simultaneous SAXS and WAXS
Where Can SAXS Help in Biology?
• Drug Discovery – Proteomics – Structural Biology
• Biochem / Biophys Basic Research
• Drug Development – Formulation – Process Control
• Biomaterials - Hair, Skin, Bone, Tissue Engineering
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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, protein-nucleic 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.
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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 ?
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SAXS in Structural Proteomics and Drug Discovery
Genomics Protein
Synthesis
Protein
Structure
Candidate
Selection
Rational
drug design
Protein sizing to determine:
• State of aggregation in solution
(monodisperse/polydisperse)?
• Suitability for crystallization?
• Effects of salts, additives, ligands?
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Biomaterials: Hair, Skin, Bone, Wood…
Small- and wide-angle patterns of oriented samples – 2D detection / sample scanning
NANOGRAPHY : This is the domain of NANOSTAR
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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
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0,0 0,1 0,2 0,3 0,4
log I
q[Å-1]
folding
atomic structure
shape
13 Å
50 Å
?
experimental data
theoretical profile from atomic model
SAXS Information on Protein Structure
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
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• Guinier plot:
• Rg, radius of gyration.
• I(0), Molecular weight
Particle Size – Guinier Law
𝐼(q) = I(0).𝑒−𝑅2q2/3
Guinier law:
Valid up to q.R ≤ 1,2
Slope = - ln(10) . R2/3
𝑙𝑛 𝐼 𝑞 = 𝑙𝑛𝐼(0) − 𝑅2 q2/3
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
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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
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dqqr
qrqqIrP
)sin(.)(
2
1)( 2
02
Fourier Transformation – The P(r) Function
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
𝑃 𝑟 = 𝑇( 𝐼(𝑞))
Limit: qmin.Dmax ≤ 𝜋
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
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Pair Distance Distribution Function P(r) Maximum particle dimension, general shape
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
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SAXS Size Range
Putnam and Hammel et al. 2007 Q R Biophysics 40:191-285
Limit:
qmin.Dmax ≤ 𝜋
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
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Solution Structure Modeling
Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org
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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
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In 1971: 10 min/point
3 min 6 min 9 min
12 min 15 min 18 min
Today:
Protein Size and Shape Within Minutes by Lab-SAXS
Stable results after < 12 min exposure
Size (Radius of Gyration) to < +/- 3 % within 3 min
Sample volume < 10 µl; concentration 0.1% -
Pair Distance Distribution
(Bruker MICROPix™)
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qmin = 0.016 Å-
1
d = 400 Å
ALD THI Lyso BSA
Speed of SAXS Dilute protein solution SAXS in human times – one coffee break
0.4%, Exposure 30 min
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SAXS Crystal Structure
Experimental vs fitted I(q)
p(R)
DIMER
Speed of Analysis – BSA model in 15 min
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• 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
Conclusions
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)
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Starting from the components:
Lipid self-assembly
Liposomes and Lipoplexes a Rich Field for SAXS/WAXS
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0.00 0.01 0.02 0.03 0.04 0.05 0.06
5.0
48.0
54.0
61.3
56.0
5.0
s [1/Å]
53.0
56.0
53.0
50.0
Thermal Phase Transitions SWAXS T-Scans
SAXS: lamellar stacking structure WAXS: hydrocarbon chain packing
Dipalmitoyl-PC (DPPC)
0.5°/min, 1 frame/min
Phases: L‘ , P‘ , L
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DSC-trace
SAXS
Drug Liposome Interaction
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Viral entry: Membrane Fusion
Viral Fusion
Protein
Viral Fusion Protein: Membrane-Peptide Screening
Searching Targets for Anti-Viral Therapy
P. Laggner, Ana J. Perez Berna and Jose Villalain, 2007
Q: Which parts of the protein are responsible for fusion?
How can viral fusion with the cell membrane be suppressed?
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FP PreTM
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 0.4
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 0.4
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 0.4
0.1 0.2 0.3 0.4
0.1 0.2 0.3 0.4
10 20 30 40 50 60 70 10 20 30 40 50 60 70 10 20 30 40 50 60 70 10 20 30 40 50 60 70 10 20 30 40 50 60 7010 20 30 40 50 60 70 10 20 30 40 50 60 70 10 20 30 40 50 60 70 10 20 30 40 50 60 70 10 20 30 40 50 60 70
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)
25°
45°
70°
q-axis (nm-1
)
197-214 309-326274-298260-277 351-368
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)
Temperature (ºC)Temperature (ºC)Temperature (ºC)
Temperature (ºC)
q-axis (nm-1
)
q-axis (nm-1
)
Temperature (ºC)
q-axis (nm-1
)
E2
E11
E13
E19
E24
Fusion Helper
0.1 0.2 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
0.1 0.2 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
0.1 0.2 0.3 0.4 0.5
0.1 0.2 0.3 0.4 0.50.1 0.2 0.3 0.4 0.50.1 0.2 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
0.1 0.2 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
0.1 0.2 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
0.1 0.2 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
0.1 0.2 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
0.1 0.2 0.3 0.4 0.5
10 20 30 40 50 60 70 10 20 30 40 50 60 70 10 20 30 40 50 60 70 10 20 30 40 50 60 70 10 20 30 40 50 60 7010 20 30 40 50 60 70 10 20 30 40 50 60 70 10 20 30 40 50 60 70 10 20 30 40 50 60 70 10 20 30 40 50 60 70
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)q-axis (nm-1
)q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm-1
)
400-417 435-452 547-564 603-620 708-725
25°
45°
70°
q-axis (nm
-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm
-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm
-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm
-1
)
q-axis (nm-1
)
q-axis (nm-1
)
q-axis (nm
-1
)
F5
F10
F27
F35
F49
Temperature (ºC)Temperature (ºC)Temperature (ºC)Temperature (ºC)
Temperature (ºC)
Host Membrane
Viral Membrane
SAXS and DSC are suitable techniques for finding regions that promote non-lamellar phases likely to be involved in fusion.
The region 603-620, fusion helper, 274-298, fusion peptide, and 309-326, PreTransmembrane domain are entry targets against HCV (hepatitis C virus).
Fusion Peptide
PreTM
Searching Targets for Anti-Viral Therapy
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‚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
‚Hybrid Analysis‘ Getting the Bigger Picture
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‚Hybrid Analysis‘ Combining Methods
SC-XRD atomic resolution structure
SAXS DSC/SAXS
structure and dynamics
NMR atomic resolution
structure in solution
Cryo-EM large-scale shape
AFM structure at surfaces
tubulin Membrane receptor
Diffraction Spectroscopy
Imaging
leads in all these fields – except for EM
MICRO-CT
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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
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SAXS – Cryo-EM : 1012 vs 1 Molecule
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
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
• 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
• NOE distance restraints: 700 x 2 • Intermolecular NOEs: 36 x 2 • Residual dipolar couplings (RDCs): 11 x 2 • RMSD 1.0 Å
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SAXS and NMR Enhanced Accuracy
NMR structure of the tetraloop receptor complex
Courtesy of Sam Butcher‘s Lab, Department of Biochemistry, University of Wisconsin
SAXS and NMR Can SAXS data substitute for intermolecular NOE and H-bond restraints?
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Courtesy of Sam Butcher‘s Lab, Department of Biochemistry, University of Wisconsin
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• NMR 25.1
• NMR+SAXS 23.1
• Measured 23.0
RG
------------------------
SAXS and NMR
Courtesy of Sam Butcher‘s Lab, Department of Biochemistry, University of Wisconsin
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
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SAXS and NMR Conclusion / Hypothesis:
divergence < 1 mrad (0.057 °)
sample rotation axis
. from multilayer optics collimator
detector
XRR
Reflection
and
refraction
Diffraction
Thin Film Structure by GISAXS
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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)
DOPC on Si chip Hecus S3-MICROpix , 1000 s
y
z
Lab-GISAXS of Phospholipid Mesophase
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DOPC/Melittin R = 10-4 in vacuo on Si
S3-MICROpix 10 min
20 hours later, air, RT
• 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
Sqashed liposomes (4,6 nm)
Surface-aligned multilayer (4.8 nm)
Functionalized Peptide – Lipid Film
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The Synchrotron Revolution SAXS Development over last 40 years
1970 1990 2010
Synchrotron
Home-lab
• Bio-SAXS, especially protein SAXS has tremendously grown with the availability of SAXS beamlines
• A major part of the synchrotron Bio-SAXS 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
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)
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The MICRO™ Revolution
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MICRO - SWAXS MICRO - SAXS
The MICRO™ - Platform SAXS system for instant laboratory nanoanalytics
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
Kratky vs Pin-Hole Collimation MICRO vs Nanostar
Defining slits Cleaning slit
Pin-hole : 360° field of view
Kratky block: 180° field of view, no background in measuring field
~500 mm
~60 mm ~300 mm
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• 1D SAXS and/or WAXS: VANTEC-1
• 2D SAXS/GISAXS: Dectris‘ Pilatus 100K
Detectors
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MICROpix – Sample Cells
• Liquid sample microcell for ambient or non-ambient
• Microcell for pastes and powders
• Capillary-flow cell for liquids and gases
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• 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)
Synchrotron-Proven Hard-and Software
• 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
S3-MICROcaliX Combining SWAX & Calorimetry*
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Microcalorimeter Microbeam X-Ray Camera
Integrated Laboratory Benchtop
System
S3-MICROcaliX
WAXS/WAXS/DSC The MICROcaliX™
60‘‘ time-frames Developed in cooperaqtion with SETARAM Caluire, France
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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
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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)
Ag-behenate powder:
20°-200°C (1.5°/min)
1D-SAXS: 1 frame/min
Speed of SAXS Calorimetric SAXS : MICROcaliX™
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Silver Behenate SAXS-DSC with Bruker MicroCaliX
ICTP School Trieste 210312
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Model of the ribbon phase
Two-dimensional centrered rectangular lattice with
space group cmm
a
b
61
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.
SAXS zoom
-10°C
38°C
Onset of transition
already at much
lower temperatures
than indicated by
calorimetry and by
WAXS
WAXS
6.4 nm
n=2
n=4 n=5
4.4. nm: second phase
Cocoa Butter: SAXS shows pre-transitional nanodomain effects
SAXS
Cocoa Butter Polymorphism – SWAXS/DSC with MICROcaliX™
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Food
Component 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 crystallization hydrocolloids melting, gelation
sugar Melting, crystallization, glass transition (amorphism)
Pharmaceutical drug, excipient Melting, polymorphism
Bio protein denaturation, aggregation, polymorphism
Lipid, membrane, vesicle Lipidic transition
Others Liquid crystal transitions
Gas hydrate Formation, dissociation
MICROcaliX™ – Fields of Application
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Innovation with Integrity
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