1. Jill Trewhella, The University of Sydney EMBO Global Exchange Lecture Course April 30, 2011 SAXS and SANS Facilities and Experiments

2. The small-angle scattering experiment FT

3. X-ray Sources  CuK  (1.54 Å) emission from Sealed X-ray tubes, practically need a line source geometry for protein work Sealed X-ray tubes, practically need a line source geometry for protein work Rotating Anode source, allows for point source geometry and hence simpler analysis Rotating Anode source, allows for point source geometry and hence simpler analysis  Synchrotrons Tunable (anomalous scattering) Tunable (anomalous scattering) High Brilliance High Brilliance Excellent for rapid scanning of conditions, very low protein concentrations, time resolved experiments, etc Excellent for rapid scanning of conditions, very low protein concentrations, time resolved experiments, etc

4. Lab-based X-ray sources

5. Point versus line collimation

6. Slit ‘smearing’

8. Anton Paar SAXSess (line source) at the University of Sydney

9. X-ray scattering samples  5-60 minute measurement times using lab based sources  Protein concentrations 1-10 mg/ml  Sample volumes in the order of 20-30 µL  Experiments using high intensity synchrotron instrumentation take a few seconds or minutes and particle concentrations can be more dilute (by at least an order of magnitude), but radiation damage can be limiting; free radical scavengers are helpful (DTT, TCEP, ascorbate)  Need a perfectly matched solvent blank; preferably a last step dialysate or column filtrate  Sample cells are made of ultra-thin quartz or mica, must be able to measure sample and solvent background in same cell, identically positioned in the beam

10. Neutron Sources  SANS requires ‘cold’ source; thermal neutrons are passed through a liquid hydrogen moderator to slow them (generally to ~4-6 Å) Reactors are steady state sources of cold neutrons that are collimated to provide a narrow wavelength band (  / ~10%) Reactors are steady state sources of cold neutrons that are collimated to provide a narrow wavelength band (  / ~10%) Spallation sources produce pulses of cold neutrons and instruments are designed to use time of flight so that all wavelengths in a given pulse can be used which compensates at least partially for relatively the low time-averaged neutron fluxes Spallation sources produce pulses of cold neutrons and instruments are designed to use time of flight so that all wavelengths in a given pulse can be used which compensates at least partially for relatively the low time-averaged neutron fluxes

11. Recall: the basic scattering equation  For an ensemble of identical, randomly oriented particles, the intensity of coherently, elastically scattered radiation is dependant only upon the magnitude of q, and can be expressed as: N = molecules/unit volume V = molecular volume = contrast, the scattering density difference = contrast, the scattering density difference between the scattering particle and solvent between the scattering particle and solvent P(q) = form factor  particle shape S(q) = structure factor  inter-particle correlation distances

12. Inter-particle distance correlations between charged molecules D D D D D - - - - - D - D - D ….. gives a non-unity S(q) term

13. Sample requirements for small-angle scattering determination of particle shape  Highly purified samples containing mono- disperse, identical particles without significant inter-particle distance correlations (S(q) = 1) Use a final gel filtration step in the purification immediately prior to measurement to eliminate any aggregates Use a final gel filtration step in the purification immediately prior to measurement to eliminate any aggregates Us DLS to evaluate samples for potential aggregates (mass fraction aggregates<0.01%) Us DLS to evaluate samples for potential aggregates (mass fraction aggregates<0.01%)

15. Essential preliminary small-angle scattering experiments  Explore the concentration dependence of the small-angle X-ray scattering to determine if S(q)  1.  If S(q)  1, adjust the solution conditions by changing pH, salt concentration, or decreasing particle concentration to eliminate  Determine the particle mass, molecular volume, and overall shapes of the components and their complex (Guinier and P(r) analyses, shape restoration)

16. Recall: Guinier Analysis  Guinier showed that a plot (lnI(q) vs q 2) gives a straight line of slope R g 2 /3 and I(0) intercept that can be interpreted in terms of the concentration, contrast and volume of the scattering particle.

17. Recall: I(q) and P(r) related by Fourier Transform Fourier transform must be done using indirect methods due to finite q-range measured; quality samples and data give well behaved transforms with certain characteristics

19. Sample Protein conc. a (mg/ml) R g (Å) D max (Å) Porod Volume b (10 3 x Å 3 ) Calculated Volume c (10 3 x Å 3 ) (10 3 x Å 3 ) MW d (kDa) SSRL data NL1-638-  (A&B) 1.8 42.5 ± 0.4 130 209 ± 20 198130/151/144 NL1/NX complex 3.6 46.8 ± 0.2 155*275181/201/199 University of Utah SAXSess instrument data NL1-638-  (A&B) 13.2 38.3 ± 0.3 7.6 40.1 ± 0.5 4.1 41.4 ± 0.6 Inf. dilution 42.4 ± 0.6 130 208 ± 14 198130/151/144 NL1-6383.3 42.7 ± 0.7 130 250 ± 19 220136/166/160 NL1-6913.8 51.8 ± 1.0 165 255 ± 26 257148/189/185 NL2-6153.7 40.6 ± 0.6 130 178 ± 7 193135/146/140 NL3-6391.2 40.3 ± 0.7 130 164 ± 12 190128/144/138 NL4-6193.4 42.1 ± 0.6 135 199 ± 7 200132/140/145 NL1/NX complex 19.7 40.9 ± 0.3 15.7 40.8 ± 0.2 9.8 44.0 ± 0.3 8.7 43.7 ± 0.4 6.6 44.5 ± 0.4 4.5 45.2 ± 0.5 3.8 47.7 ± 0.9 155* Inf. dilution 47.7 ± 0.8 155*275181/201/199

20. 265,851228,139 ± 9,96547.1 ± 0.7 Complex 199,26142.19 ± 0.7 NL1-638-Δ(A&B) Vol (Å 3 ) Calculated Vol (Å 3 ) ExperimentalRg (Å)Sample 184,172 ± 7,778

21. Determining the size of your scattering particle  Place data on an absolute scale (water scattering) and use: Orthaber et al. (2000) J. Appl. Cryst. 33, 218  Use a known mono-disperse protein scatterer (such as lysozyme) and use: Krigbaum and Kugler (1970) Biochemistry 9, 1216 Krigbaum and Kugler (1970) Biochemistry 9, 1216

22. Fischer et al. (2010) The molecular weight of proteins in solution can be determined from a single SAXS measurement on a relative scale. J. Appl. Cryst. 43,101 If you scale your data so that I(0) = 1, then: where where In practice we can only calculate: So Fischer et al calculated correction factors to relate the ‘apparent’ volume using Q’ to the actual volume based on 1148 unique, known structures and their model profiles.

23. Planning the neutron scattering experiment  Choose your data collection strategy (solvent matching or contrast variation?)  Determine how much sample is needed  Decide which subunit to label  What deuteration level is needed in the labeling subunit   See MULCh* http://www.mmb/usyd.edu.au/NCVWeb/ *MULCh, Whitten et al, accepted J. Appl. Cryst. 2007

24. MULCh  ModULes for the analysis of neutron Contrast variation data Contrast, computes neutron contrasts of the components of a complex Contrast, computes neutron contrasts of the components of a complex R g, analyses the contrast dependence of the radius of gyration to yield information relating to the size and disposition of the labelled and unlabeled components in a complex R g, analyses the contrast dependence of the radius of gyration to yield information relating to the size and disposition of the labelled and unlabeled components in a complex Compost, decomposes the contrast variation data into composite scattering functions containing information on the shape of the lab\led and unlabeled components and their dispositions Compost, decomposes the contrast variation data into composite scattering functions containing information on the shape of the lab\led and unlabeled components and their dispositions

25. Solvent matching  Best used when you are interested in the shape of one component in a complex, possibly how it changes upon ligand binding or complex formation.  Requires enough of the component to be solvent matched to complete a contrast variation series to determine required %D 2 O (~4 x 200-300  L, ~5 mg/ml).  Requires 200-300  L of the labeled complex at 5-10mg/ml.

26. Solvent Match Point Determination

27. Apical view Front view Side view 90°

28. Co-refinement of the  neurexin positions and orientations with respect to NL1 give a model against the X-ray and neutron data gives us a model that we can map autism-linked mutations R451C V403M K378R G99S Comoletti, Grishaev, Whitten et al. Structure 15, 693-705, 2007.

29. Superposition of solution scattering and crystal structure for NL-NX

30. Contrast variation  To determine the shapes and dispositions of labeled and unlabelled components in a complex  Requires  5 x 200-300  L (= 1 – 1.5mL) of your labeled complex at  5 mg/ml.  Deuteration level in labeled protein depends upon its size. Smaller components require higher levels of deuteration to be distinguished. Smaller components require higher levels of deuteration to be distinguished. Ideally would like to be able to take data at the solvent match points for the labeled and unlabeled components Ideally would like to be able to take data at the solvent match points for the labeled and unlabeled components

31.  Measure sample and solvent blanks at each contrast point (use a broad range of D 2 O concentrations; e.g. 0,20,40, 80, 100% D 2 O)  Subtract solvent blank data from sample  Sample to low-q with sufficient frequency to determine large distances accurately (min. 15-20 points in the Guinier region)  Measure to high enough q to aid in checking background subtraction (q = 0.45 Å -1 )  q = 0.01 -.45 is typical range for 10- 150 kDa particles, usually requires two detector positions

32. Effects of incoherent scattering from 1 H on backgrounds  HCaM measurement was done in 42% D 2 O to solvent match the HCaM. Objective was to see DCaM in presence of HCaM, but without interference from HCaM  Incoherent scattering from 1 H is a constant with q

33.  X-ray scattering data from LacI, with insert showing Guinier plot with adequate sampling.

34. Use Rg (from MULCh) for Sturhman analysis R KinA = 25.40 Å R Sda = 25.3 Å D = 27.0 Å

35. Use Compost (from MULCh) to solve for I(q) 11, I(q) 22, I(q) 12 I1I1I1I1 I 12 I2I2I2I2

36. Use SASREF7 to do rigid body refinement of the components against the scattering data (if you have pdb files for components)  2 = 1.27  2 = 0.97  2 = 0.63  2 = 0.56  2 = 0.76  2 = 0.92  2 = 1.12  2 = 0.95 22

37. Incorporation of deuterium up to 86% of the chemically Non-exchangeable protons can be obtained by using D 2 O as the deuterium source. Complete deuteration can only be obtained by addition of perdeuterated carbon source (glucose or glycerol). Use mass spec to determine deuteration levels. The described protocols allow the deuteration content in recombinant proteins to be predicted

38. Neutron scattering sample cells  Helma quartz cells (high precision path-length, suprasil) – need lots of them!  Banjo-style (280  L per 1 mm path length) or rectangular (170  L per 1 mm path length) cells can be used  Path lengths are only good to 1%, so good idea to measure sample and solvent background in the same cell if practical, but experiment logistics may prohibit that, so often have to ‘fudge’ background subtractions  High incoherent scattering for 1 H means you always want  1mm 1 H 2 O in the neutron beam to avoid multiple scattering

39. Doing a Quality Experiment   After your final gel filtration step, check out your samples with dynamic light scattering   Carefully calibrate your concentration assay – colorimetric assays are almost useless, extinction coefficient is good if strong enough, quantitative amino acid analysis can work   Compare your data to a well characterized standard(s)   For protein/DNA complexes, standards are more difficult. Measure the partial specific volume of your particle if you have enough sample – or use a good model to calculate it, e.g. see MULCh or http://geometry.molmovdb.org/NucProt/

40. Neutrons  Non-ionizing radiation  Penetrating  Wavelength and energies available that are suitable for probing structures with dimensions 1-1000s Å  Coherent scattering lengths that vary randomly with atomic weight and large isotope effect for hydrogen – contrast variation  Large incoherent scattering cross-section for 1 H is a source of noise in small-angle scattering  Interact weakly with matter and are difficult to produce and detect – therefore should only be used when they provide information that cannot be otherwise obtained.

41. Assessing the quality of small-angle scattering results  Are there instrumental effects unaccounted for?  Are the scattering particles mono-disperse and identical or is there a conformational ensemble?  Do you have dilute solution conditions?  Do the data show the expected Guinier and Porod behavior?  Is the P(r) “well-behaved?”  Are background subtractions accurate?  Have standards been measured?  How well characterized is the sample (purity, concentration)  Are errors appropriately handled – can you rely on  2 ?

42. Jacques & Trewhella (2010) “Small-angle Scattering for Structural Biology; Expanding the Frontier While Avoiding the Pitfalls,” Protein Science 19, 642-657