SAXS and SANS Facilities and Experiments Jill Trewhella

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SAXS and SANS Facilities and Experiments Jill Trewhella, The University of Sydney EMBO Global

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

The small-angle scattering experiment FT

The small-angle scattering experiment FT

X-ray Sources Ø Cu. K (1. 54 Å) emission from l l Sealed X-ray

X-ray Sources Ø Cu. K (1. 54 Å) emission from l l 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 Ø Synchrotrons l l l Tunable (anomalous scattering) High Brilliance Excellent for rapid scanning of conditions, very low protein concentrations, time resolved experiments, etc

Lab-based X-ray sources

Lab-based X-ray sources

Point versus line collimation

Point versus line collimation

Slit ‘smearing’

Slit ‘smearing’

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

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

X-ray scattering samples Ø Ø Ø 5 -60 minute measurement times using lab based

X-ray scattering samples Ø Ø Ø 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

Neutron Sources Ø SANS requires ‘cold’ source; thermal neutrons are passed through a liquid

Neutron Sources Ø SANS requires ‘cold’ source; thermal neutrons are passed through a liquid hydrogen moderator to slow them (generally to ~4 -6 Å) l l 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

Recall: the basic scattering equation ØFor an ensemble of identical, randomly oriented particles, the

Recall: the basic scattering equation ØFor 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 between the scattering particle and solvent P(q) = form factor particle shape S(q) = structure factor inter-particle correlation distances

Inter-particle distance correlations between charged molecules D - - D D D - ….

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

Sample requirements for small-angle scattering determination of particle shape Ø Highly purified samples containing

Sample requirements for small-angle scattering determination of particle shape Ø Highly purified samples containing mono- disperse, identical particles without significant inter-particle distance correlations (S(q) = 1) l l 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%)

Essential preliminary small-angle scattering experiments Explore the concentration dependence of the small-angle X-ray scattering

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

Recall: Guinier Analysis Ø Guinier showed that a plot (ln. I(q) vs q 2)

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

Recall: I(q) and P(r) related by Fourier Transform Fourier transform must be done using

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

Sample Protein conc. a (mg/ml) Rg (Å) NL 1 -638 - (A&B) 1. 8

Sample Protein conc. a (mg/ml) Rg (Å) NL 1 -638 - (A&B) 1. 8 42. 5 ± 0. 4 NL 1/NX complex 3. 6 46. 8 ± 0. 2 Porod Volumeb (103 x Å3) Calculated Volumec (103 x Å3) MWd (k. Da) 130 209 ± 20 198 130/151/144 155 * 275 181/201/199 Dmax (Å) SSRL data University of Utah SAXSess instrument data NL 1 -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 ± 14 198 130/151/144 NL 1 -638 3. 3 42. 7 ± 0. 7 130 250 ± 19 220 136/160 NL 1 -691 3. 8 51. 8 ± 1. 0 165 255 ± 26 257 148/189/185 NL 2 -615 3. 7 40. 6 ± 0. 6 130 178 ± 7 193 135/146/140 NL 3 -639 1. 2 40. 3 ± 0. 7 130 164 ± 12 190 128/144/138 NL 4 -619 3. 4 42. 1 ± 0. 6 135 199 ± 7 200 132/140/145 NL 1/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 * 275 181/201/199

P(r) function comparison between NL 1 and its complex with NX Sample Rg (Å)

P(r) function comparison between NL 1 and its complex with NX Sample Rg (Å) Vol (Å3) Experimental Calculated 55 50 45 Complex 47. 1 ± 0. 7 228, 139 ± 9, 965 265, 851 NL 1 -638 -Δ(A&B) 42. 19 ± 0. 7 184, 172 ± 7, 778 199, 261 P(r) arbitrary units 40 Complex NL 1 -638 -Δ(A&B) 35 30 25 20 15 10 5 0 0 10 20 30 40 50 60 70 80 90 100 110 120130 140 150 Distance (Angstroms)

Determining the size of your scattering particle Ø Place data on an absolute scale

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

Fischer et al. (2010) The molecular weight of proteins in solution can be determined

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 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.

Planning the neutron scattering experiment Ø Choose your data collection strategy (solvent matching or

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

MULCh Ø Mod. ULes for the analysis of neutron Contrast variation data l l

MULCh Ø Mod. ULes for the analysis of neutron Contrast variation data l l l Contrast, computes neutron contrasts of the components of a complex Rg, 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 labled and unlabeled components and their dispositions

Solvent matching Ø Best used when you are interested in the shape of one

Solvent matching Ø 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. Ø 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). Ø Requires 200 -300 L of the labeled complex at 5 -10 mg/ml.

Solvent Match Point Determination

Solvent Match Point Determination

Front view 90° Side view 90° Apical view

Front view 90° Side view 90° Apical view

G 99 S Co-refinement of the neurexin positions and orientations with respect to NL

G 99 S Co-refinement of the neurexin positions and orientations with respect to NL 1 give a model against the X-ray and neutron data gives us a model that we can map autism-linked mutations K 378 R R 451 C V 403 M Comoletti, Grishaev, Whitten et al. Structure 15, 693 -705, 2007.

Superposition of solution scattering and crystal structure for NL-NX

Superposition of solution scattering and crystal structure for NL-NX

Contrast variation To determine the shapes and dispositions of labeled and unlabelled components in

Contrast variation To determine the shapes and dispositions of labeled and unlabelled components in a complex Ø Requires 5 x 200 -300 L (= 1 – 1. 5 m. L) of your labeled complex at 5 mg/ml. Ø Deuteration level in labeled protein depends upon its size. Ø l l 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

Ø Ø Ø Measure sample and solvent blanks at each contrast point (use a

Ø Ø Ø 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 10150 k. Da particles, usually requires two detector positions

Effects of incoherent scattering from 1 H on backgrounds HCa. M measurement was done

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

Ø X-ray scattering data from Lac. I, with insert showing Guinier plot with adequate

Ø X-ray scattering data from Lac. I, with insert showing Guinier plot with adequate sampling.

Use Rg (from MULCh) for Sturhman analysis RKin. A = 25. 40 Å RSda

Use Rg (from MULCh) for Sturhman analysis RKin. A = 25. 40 Å RSda = 25. 3 Å D = 27. 0 Å

Use Compost (from MULCh) to solve for I(q)11, I(q)22, I(q)12 I 2

Use Compost (from MULCh) to solve for I(q)11, I(q)22, I(q)12 I 2

Use SASREF 7 to do rigid body refinement of the components against the scattering

Use SASREF 7 to do rigid body refinement of the components against the scattering data (if you have pdb files for components) 2 2 = 1. 27 2 = 0. 97 2 = 0. 63 2 = 0. 56 2 = 0. 76 2 = 0. 92 2 = 1. 12 2 = 0. 95

The described protocols allow the deuteration content in recombinant proteins to be predicted Incorporation

The described protocols allow the deuteration content in recombinant proteins to be predicted 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.

Neutron scattering sample cells Helma quartz cells (high precision path-length, suprasil) – need lots

Neutron scattering sample cells Helma quartz cells (high precision path-length, suprasil) – need lots of them! Ø Banjo-style (280 L per 1 mm path length) or rectangular (170 L per 1 mm path length) cells can be used Ø 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 Ø High incoherent scattering for 1 H means you always want 1 mm 1 H 2 O in the neutron beam to avoid multiple scattering Ø

Doing a Quality Experiment After your final gel filtration step, check out your samples

Doing a Quality Experiment After your final gel filtration step, check out your samples with dynamic light scattering Ø Carefully calibrate your concentration assay – colorimetric assays are almost useless, extinction coefficient is good if strong enough, quantitative amino acid analysis can work Ø Compare your data to a well characterized standard(s) Ø 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/Nuc. Prot/ Ø

Neutrons Ø Ø Ø Non-ionizing radiation Penetrating Wavelength and energies available that are suitable

Neutrons Ø Ø Ø Non-ionizing radiation Penetrating Wavelength and energies available that are suitable for probing structures with dimensions 1 -1000 s 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.

Assessing the quality of small-angle scattering results Ø Ø Ø Ø Ø Are there

Assessing the quality of small-angle scattering results Ø Ø Ø Ø Ø 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?

Jacques & Trewhella (2010) “Small-angle Scattering for Structural Biology; Expanding the Frontier While Avoiding

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