Bruker AXS Overview of Biological SAXS Webinar 20120614

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 

X-Ray Beam 

> 6° 

XRD/WAXS 

Internal structure, 

Crystal parameters 

0.02 - 6° 

SAXS 

Particle size/shape 

Nanosized pores, 

Defects 

5

The Length Scales 

x 10 6 

nm µm mm 

SWAXS 

Liposomal 

Carrier 

Porous mineral 

The 

Product 

This is where all the 

chemistry happens 

This is our reality 

6

Typical Time Scales 

-6 -4 -2 0 2 

log t (s) 

Synchrotron beamlines 

Lab instruments 

7

The Reciprocity: Size – Scattering Angle 

Small 

particles 

Wide SAXS range 

Large 

particles 

Narrow SAXS range 

8

angle (degrees) 

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 

40 

WAXS 

30 

Molecular Lattice 

Unit cell dimensions 

symmetry 

20 

10 

0 

Nano-Envelope 

Size / Shape 


10 100 1000 

distance (A) 

9

Advantages of Simultaneous 

SAXS and WAXS 



crystalline 

Scattering 

curve 

amorphous 

150 nm 1 

10 Angström 3 

Nanoscale 

domains/particles/ 

pores 

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 (R G ) , molecular weight , max. 

dimension 

Q: Search for optimum crystallization conditions 

‣ SAXS in different salt or polymer solutions – size (R G ) 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

log I 

SAXS Information on Protein Structure 

shape 

experimental data 

theoretical profile from atomic model 

 

folding 

atomic structure 

50 Å 

0,0 0,1 0,2 0,3 0,4 

Graphics courtesy of Michal Hammel SAXS Consulting www.saxs.org 

q[Å -1 ] 

13 Å 

17

Particle Size – Guinier Law 

Guinier law: 

I(q) = I(0).e −R2 q 2 /3 

ln I q = lnI(0) − R 2 q 2 /3 

Valid up to q.R ≤ 1,2 

• Guinier plot: 

• Rg, radius of gyration. 

• I(0), Molecular weight 

Slope = - ln(10) . R 2 /3 


18

Protein Folding - Kratky Plot 

The appearance of the 

SAXS curve in the 

Kratky plot allows to 

infer chain folding 

characteristics - 


19

Fourier Transformation – The P(r) Function 

P( 

r) 

 

1 

2 

 

2 

 

0 

I( 

q). 

q 

2 

sin( qr) 

qr 

dq 

Limit: 

q min .D max ≤ 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 


20

Pair Distance Distribution Function P(r) 

Maximum particle dimension, general shape 


21

SAXS Size Range 

Limit: 

q min .D max ≤ π 

Putnam and Hammel et 

al. 2007 Q R Biophysics 

40:191-285 


22

Solution Structure Modeling 


23

Proteomic Scale, Shape and Assembly 

Hura, Menon, Hammel et al. 

(2009) Nature Methods 6, 

606 - 61 


24

In 1971: 10 min/point 

25

Today: 

Protein Size and Shape Within Minutes by Lab-SAXS 

3 min 6 min 9 min 

Pair Distance 

Distribution 

12 min 15 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 

q min = 0.016 Å - 

1 

d = 400 Å 

27

Speed of Analysis – BSA model in 15 min 

Experimental vs 

fitted I(q) 

Crystal Structure 


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 

Dipalmitoyl-PC (DPPC) 

0.5°/min, 1 frame/min 

Phases: L ‘ , P ‘ , L 

32 

SAXS: lamellar stacking structure 

WAXS: hydrocarbon 

chain packing 

5.0 

48.0 

50.0 

53.0 

54.0 

61.3 56.0 

56.0 

53.0 

5.0 

0.00 0.01 0.02 0.03 0.04 0.05 0.06 

s [1/Å]

Drug Liposome Interaction 

DSC-trace 


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: Membrane- 

Peptide Screening 

Viral entry: 

Membrane 

Fusion 

Viral Fusion 

Protein 

P. Laggner, Ana J. Perez Berna and Jose Villalain, 2007 

34

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 70 

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 70 

Host Membrane 

Viral Membrane 

197-214 260-277 274-298 

309-326 

351-368 

400-417 435-452 547-564 603-620 708-725 

25° 

25° 

q-axis (nm -1 ) 










45° 

45° 











70° 

70° 








E2 

E11 

FP 



PreTM 

E13 E19 E24 


F5 

F10 

Fusion Helper 

F27 F35 F49 

Temperature (ºC) 




Temperature (ºC)

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 

SC-XRD 

atomic resolution structure 

Spectroscopy 


DSC/SAXS 

structure and 

dynamics 

Membrane receptor 

tubulin 

NMR 

atomic resolution 

structure in solution 

Cryo-EM 

large-scale shape 

Imaging 

AFM 

structure at surfaces 

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 : 10 12 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


Can SAXS data substitute for intermolecular NOE and H-bond restraints 


42


R G 

------------------------ 

• NMR 25.1 

• NMR+SAXS 23.1 

• Measured 23.0 


43


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 

Diffraction 

Reflection 

and 

refraction 

XRR 

detector 

from multilayer optics 

collimator 

. 

sample rotation axis 

divergence < 1 mrad (0.057 °) 

45

Lab-GISAXS of Phospholipid Mesophase 

z 

DOPC on Si chip 

Hecus S3-MICROpix , 1000 s 

y 

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 

S3-MICROpix 10 min 

20 hours later, air, RT 

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 

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 

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: 

True SAXS : 

Interactive : 

3 x higher flux than other lab-SAXS system by IµS-microsource 

(INCOATEC) and MICRO Kratky-optics* 

No information loss through point-beam focusing / no de-smearing 

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 

Cleaning slit 

~500 mm 

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

Combining SWAX S3-MICROcaliX & 

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 

S3-MICROcaliX 

Microcalorimeter 

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 Polymorphism – 

SWAXS/DSC with MICROcaliX 

Cocoa Butter: SAXS shows pre-transitional nanodomain effects 


6.4 nm 

4.4. nm: second phase 

n=2 

38°C 

n=4 n=5 

-10°C 

SAXS zoom 

Onset of transition 

already at much 

lower temperatures 

than indicated by 

calorimetry and by 



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 

Food 

Component 

protein 

Protein powder 

starch 

milk 

fat, chocolate 

fat 

hydrocolloids 

sugar 

Application 

denaturation, aggregation 

crystallization 

gelatinisation, retrogradation, glass 

transition 

Melting, crystallization, denaturation, 

aggregation 

Melting, crystallization, lipidic transition, 

polymorphism 

crystallization 

melting, gelation 

Melting, crystallization, glass transition 

(amorphism) 

Pharmaceutical drug, excipient Melting, polymorphism 

Bio 

Others 

protein 

Lipid, membrane, vesicle 

Liquid crystal 

Gas hydrate 

denaturation, aggregation, polymorphism 

Lipidic transition 

transitions 

Formation, dissociation 

63

Innovation with Integrity 

© Copyright Bruker Corporation. All rights reserved. 64

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