1. Macromolecular Small-Angle Scattering with Synchrotron Radiation Tom Irving BioCAT, Dept. BCPS and CSRRI Illinois Institute of Technology

2. Scope of Lecture Why do SAXS? Physical Principles Experimental methods Data interpretation Advantages of Third Generation Synchrotrons for SAXS References for learning more

3. What is SAXS? Small Angle X-ray Scattering Scattering proportional to /Molecular size Typical x-ray wavelengths ~ 0.1 nm Typical molecular dimensions 1 -100 nm Scattering angles are small 0-2 o historically. Now 0-15 o range is of increasing experimental interest

4. Why SAXS ? Atomic level structures from crystallography or NMR “gold standard” for structural inferences Crystallography, by definition, studies static structures Most things crystallize only under rather specific, artificial conditions Kinetics of molecular interactions frequently of interest SAXS can provide useful, although limited, information on relatively fast time scales

5. What is SAXS Used for? Estimating sizes of particulates Interactions in fluids Sizes of micelles etc in emulsions Size distributions of subcomponents in materials Structure and dynamics of biological macromolecules

6. SAXS and Biological Macro- molecules How well does the crystal structure represents the native structure in solution? Can we get even some structural information from large proportion of macro-molecules that do not crystallize? How can we test hypotheses concerning large scale structural changes on ligand binding etc. in solution SAXS can frequently provide enough information for such studies May even be possible to deduce protein fold solely from SAXS data

7. Scattering from Molecules Molecules are much larger than the wavelength (~0.1 nm) used => scattered photons will differ in phase from different parts of molecule Observed intensity spherically averaged due to molecular tumbling e-e- e-e- e-e- e-e- e-e- e-e- Constructive interference destructive interference

8. Intensity in SAXS Experiments: Sum over all scatterers (electrons) in molecule to get structure factor (in units of scattering 1 electron) F(q) =  i e i q ri Intensity is square (complex conjugate) of structure factor I(q) = F F* =  j  i e iq r i,j Isotropic, so spherical average (  is rotation angle relative to q) I(q) =   j  i e I q r i,j sin  d  Debye Eq. (q) =  i  j sin q r i,j / q r i,j where q = 4  sin  /

9. In Scattering Experiments, Particles are Randomly Oriented Intensity is spherically averaged Phase information lost Low information content fundamental difficulty with SAXS Only a few, but frequently very useful, structural parameters can be unambiguously obtained.

10. Structural Parameters Obtainable from SAXS Molecular weight* Molecular volume* Radius of gyration (Rg) Distance distribution function p( r ) Various derived parameters such as longest cord from p ( r ) * requires absolute intensity information

11. Experimental Geometry 200 cm 30 cm “long camera ~1 o short camera ~ 15 o Detector Sample in 1 mm capillary Collimated X-ray beam Backstop

12. The data: Shadow of lead beam stop 2-D data needs to be radially integrated to produce 1-D plots of intensity vs q

13. Scattering Curves From Cytochrome C q nm -1 ln I Red line = sample +buffer Blue = buffer only Black = difference I

14. What does this look like for a typical protein ? Since a Fourier transform, inverse relationship: Large features at small q Small features at large q Globular size 2 o structure Domain folds

15. What’s Rg? Analogous to moment of inertia in mechanics Rg 2 =  p(r)  r 2  dV  p(r)  dV

16. Rg for representative shapes Sphere Rg 2 = 3/5r 2 Hollow sphere (r1 and r2 inner and outer radii) Rg 2 = 3/5 (r2 5 -r1 5 )/(r2 3 -r1 3 ) Ellipsoid (semi-axis a, b,c) Rg 2 = (a 2 +b 2 +c 2 )/5

17. Estimating Molecular Size from SAXS Data (  ) =  i  j sin q r i,j / qr i,j Taylor series expansion = 1 - (qr ij ) 2 /6 + (qr ij ) 4 /120 …. Guinier approximation: e -q 2 Rg 2 /3 = 1 – q 2 R g 2 /3 + (q 2 R g 2 /3 ) 2 /2! … Equate first two terms 1 - (qr ij ) 2 /6 = 1 – q 2 R g 2 3 Or ln I/I0 = q 2 R g 2 /3

18. Guinier Plot Plot ln I vs. q 2 Inner part will be a straight line Slope proportional to Rg 2 –Only valid near q = 0 (i.e. where third term is insignificant) –For spherical objects, Gunier approximation holds even in the third term… so the Guinier region is larger for more globular proteins –Usual limit: R g q max <1.3

19. Configuration Changes in Plasminogen EACA Bz

20. PgRg PBS 30.6 +EACA 49.1 +Benzamidine 37.1 Guinier Fits Plasminogen data courtesy N. Menhart IIT

21. Need for Series of Concentrations SAXS intensity equations valid only at infinite dilution Excess density of protein over H 2 O very low Need a non-negligible concentration ( > 1 mg/ml) to get enough signal. In practice use a concentration series from ~ 3 - 30 mg/ml and extrapolate to zero by various means Only affects low angle regime Can use much higher concentrations for high angle region (where scattering weak anyway)

22. Effect of Concentration

23. Correcting for Concentration

24. Shape information SAXS patterns have relatively low information content Sources of information loss: –Spherical averaging –X-ray phase loss, so can’t invert Fourier transform In general cannot recover full shape, but can unambiguously compute distribution of distance s within molecule: i.e. p(r) function

25. p(r) Distribution of distances of atoms from centroid Autocorrelation function of the electron density 1-D: Only distance, not direction –No phase information –Can be determined unambiguously from SAXS pattern if collected over wide enough range –20:1 ratio qmin :qmax usually ok e-e- e-e- e-e- e-e- e-e-

26. Relation of p( r ) to Intensity I(q) = 4   0 D p( r )sin  qr  dr

27. Relationship of shape to p(r) Fourier transform pair p(r)  I(q) shape Can unambiguously calculate p( r ) from a given shape but converse not true

28. Inversion intensity equation not trivial Need to worry about termination effects, experimental noise and various smearing effects Inversion of intensity equation requires use of various “regularization approaches” One popular approach implemented in program GNOM (Svergun et al. J. Appl. Cryst. 25:495)

29. Example of p(r ) Analysis

30. Troponin C structure Does p(r) make sense?

31. Scattering Pattern from Troponin C q nm -1 I

32. Troponin C: Bimodal Distribution

33. Hypothesis Testing with SAXS p (r ) gives an alternative measure of Rg and also “longest cord” Predict Rg and p( r ) from native crystal structure (tools exist for pdb data) and from computer generated hypothetical structures under conditions of interest Are the hypothesized structures consistent with SAXS data?

34. SAXS Data Alone Cannot Yield an Unambiguous Structure One can combine Rg and P( r ) information with: Simulations based on other knowledge (i.e. partial structures by NMR or X-ray) Or Whole pattern simulations using various physical criteria: –Positive e density, –finite extent, –Connectivity –chemically meaningful density distributions

35. Reconstruction of Molecular Envelopes Very active area of research 3 main approaches: Spherical harmonic-based algorithms (Svergun, & Stuhrmann,1991, Acta Crystallogr. A47, 736), genetic algorithms (Chacon et al, 1998, Biophys. J. 74, 2760), simulated annealing (Svergun,1999Biophys. J. 76, 2879), and “give ‘n take” algorithms (Walter et al, 2000, J. Appl. Cryst 33, 350). Latter three make use of “Dummy atom approach” using the Debye formula.

36. Configuration Changes in Plasminogen EACA Bz

37. PgRg PBS 30.6 +EACA 49.1 +Benzamidine 37.1 Guinier Fits Plasminogen data courtesy N. Menhart IIT

38. Pg Complete Scattering curves

39. +EACA+BNZ 2Å2Å 2Å2Å Shape Reconstruction using SAXS3D *: * D. Walther et. al., UCSF

40. Technical Requirements for SAXS Monodispersed sample (usually) Very stable, very well collimated beam Very mechanically stable apparatus Methods to assess and control radiation damage and radiation induced aggregation (flow techniques) Ability to accurately measure and correct for variations in incident and transmitted beam intensity High dynamic range, high sensitivity and low noise detector

41. Detectors For SAXS 1-D or 2 D position sensitive gas proportional counters –Pros: High dynamic range, zero read noise –Cons: limited count rate capability typically 10 5 - 10 6 cps, 1-D detectors very inefficient high q range 2D CCD detectors –Pros: integrating detectors - no intrinsic count rate limit, 2-D so can efficiently collect high q data –Cons: Significant read noise, finite dynamic range –Most commercial detectors designed for crystallography too high read noise

42. SAXS at Third Generation Synchrotron Sources

43. The Advanced Photon Source

44. The APS is Optimized for Producing Undulator Radiation

45. Why is APS Undulator Radiation Good for Biological Studies? Wide energy range available for spectroscopy High flux for time resolved applications Very low beam divergence for high quality diffraction/scattering patterns Can focus to very small beams to examine small samples or regions within samples

46. What is BioCAT? A NIH-supported research center for the study of partially ordered and disordered biological materials Supported techniques are X-ray Spectroscopy (XAS and high resolution), powder diffraction, fiber diffraction, and SAXS Comprises an undulator based beamline, (18-ID) associated laboratory and computational facilities. Available to all scientists on basis of peer-reviewed beamtime proposals

47. The BioCAT Sector at the APS

48. SAXS Instrument on the BioCAT 18ID - Undulator Beamline

50. BioCAT PERFORMANCE FOR SAXS 3 m camera can access a range of q from ~0.04 to 1.3 nm -1 0.3 m camera accesses range of q from ~0.8 to 20.0 nm -1 55 x 88 mm high sensitivity CCD detector can detect single photons Useful SAXS patterns can be collected from 5 mg./ml cytochrome c in 300 ms => can do time resolved experiments on ms time scales or less

51. Why Do You Need a Third Generation Source for SAXS? Time resolved protein folding studies using SAXS => The “Protein Folding Problem” High throughout molecular envelope determinations using SAXS => “Structural genomics”

52. Time-resolved Stopped-flow Experiment Time-resolve Stopped Flow Experiment

53. For further reading….. A Guinier “X-ray Diffraction in Crsytals, Imperfect Crystals and Amorphous Bodies” Freeman, 1963 C. Cantor and P. Schimmel “Biophysical Chemistry part II: Techniques for the study of Biological Strcutre and Function” Freeman, 1980 O. Glatter and O. Kratky “Small-angle X-ray Scattering” Academic Press 1982 See Dmitri Svergun’s web site at http://www.embl- hamburg.de/Externalinfo/Research/Sax http://www.embl- hamburg.de/Externalinfo/Research/Sax