Jill Trewhella, The University of SydneyJill Trewhella, The University of SydneyEMBO Global Exchange Lecture CourseEMBO Global Exchange Lecture Course

April 30, 2011April 30, 2011

SAXS and SANS Facilities and ExperimentsSAXS and SANS Facilities and Experiments

The small-angle scattering experiment

FT

X-ray Sources X-ray Sources

CuKCuK (1.54 Å) emission from (1.54 Å) emission from Sealed X-ray tubes, practically need a line Sealed X-ray tubes, practically need a line

source geometry for protein worksource geometry for protein work Rotating Anode source, allows for point source Rotating Anode source, allows for point source

geometry and hence simpler analysisgeometry and hence simpler analysis SynchrotronsSynchrotrons

Tunable (anomalous scattering)Tunable (anomalous scattering) High BrillianceHigh Brilliance Excellent for rapid scanning of conditions, very Excellent for rapid scanning of conditions, very

low protein concentrations, time resolved low protein concentrations, time resolved experiments, etcexperiments, etc

Lab-based Lab-based X-ray sourcesX-ray sources

Point versus line collimationPoint versus line collimation

Slit ‘smearing’Slit ‘smearing’

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

X-ray scattering samplesX-ray scattering samples 5-60 minute measurement times using lab based sources 5-60 minute measurement times using lab based sources Protein concentrations 1-10 mg/mlProtein concentrations 1-10 mg/ml Sample volumes in the order of 20-30 µLSample volumes in the order of 20-30 µL Experiments using high intensity synchrotron Experiments using high intensity synchrotron

instrumentation take a few seconds or minutes and particle instrumentation take a few seconds or minutes and particle concentrations can be more dilute (by at least an order of concentrations can be more dilute (by at least an order of magnitude), but radiation damage can be limiting; free magnitude), but radiation damage can be limiting; free radical scavengers are helpful (DTT, TCEP, ascorbate)radical scavengers are helpful (DTT, TCEP, ascorbate)

Need a perfectly matched solvent blank; preferably a last Need a perfectly matched solvent blank; preferably a last step dialysate or column filtrate step dialysate or column filtrate

Sample cells are made of ultra-thin quartz or mica, must be Sample cells are made of ultra-thin quartz or mica, must be able to measure sample and solvent background in same able to measure sample and solvent background in same cell, identically positioned in the beam cell, identically positioned in the beam

Neutron SourcesNeutron Sources SANS requires ‘cold’ source; thermal neutrons SANS requires ‘cold’ source; thermal neutrons

are passed through a liquid hydrogen are passed through a liquid hydrogen moderator to slow them (generally to ~4-6 Å) moderator to slow them (generally to ~4-6 Å) Reactors are steady state sources of cold neutrons Reactors are steady state sources of cold neutrons

that are collimated to provide a narrow wavelength that are collimated to provide a narrow wavelength band (band (// ~10%) ~10%)

Spallation sources produce pulses of cold neutrons Spallation sources produce pulses of cold neutrons and instruments are designed to use time of flight and instruments are designed to use time of flight so that all wavelengths in a given pulse can be so that all wavelengths in a given pulse can be used which compensates at least partially for used which compensates at least partially for relatively the low time-averaged neutron fluxes relatively the low time-averaged neutron fluxes

Recall: the basic scattering equationRecall: the basic scattering equation

For an ensemble of identical, randomly oriented For an ensemble of identical, randomly oriented particles, the intensity of coherently, elastically particles, the intensity of coherently, elastically scattered radiation is dependant only upon the scattered radiation is dependant only upon the magnitude of magnitude of qq, and can be expressed as:, and can be expressed as:

N N = molecules/unit volume= molecules/unit volumeV V = molecular volume= molecular volume = contrast, the scattering density difference = contrast, the scattering density difference

between the scattering particle and solventbetween the scattering particle and solventP(q)P(q) = form factor = form factor particle shape particle shape S(q)S(q) = structure factor = structure factor inter-particle correlation distances inter-particle correlation distances

)()()( 2 qSqPVNqI

s )(r

Inter-particle distance correlations Inter-particle distance correlations between charged molecules between charged molecules

D

D

D

D

D

-

-

--

-

D

-

D

-

D

….. gives a non-unity S(q) term

Sample requirements for small-angle scattering Sample requirements for small-angle scattering determination of particle shapedetermination of particle shape

Highly purified samples containing mono-Highly purified samples containing mono-disperse, identical particles without disperse, identical particles without significant inter-particle distance significant inter-particle distance correlations (correlations (SS((qq) = 1)) = 1) Use a final gel filtration step in the purification Use a final gel filtration step in the purification

immediately prior to measurement to eliminate immediately prior to measurement to eliminate any aggregatesany aggregates

Us DLS to evaluate samples for potential Us DLS to evaluate samples for potential aggregates (mass fraction aggregates (mass fraction aggregates<0.01%)aggregates<0.01%)

Essential preliminary Essential preliminary small-angle scattering experimentssmall-angle scattering experiments

Explore the concentration dependence of the Explore the concentration dependence of the small-angle X-ray scattering to determine if small-angle X-ray scattering to determine if SS((qq) ) 1. 1.

If If SS((qq) ) 1, adjust the solution conditions by 1, adjust the solution conditions by changing pH, salt concentration, or decreasing changing pH, salt concentration, or decreasing particle concentration to eliminateparticle concentration to eliminate

Determine the particle mass, molecular volume, Determine the particle mass, molecular volume, and overall shapes of the components and their and overall shapes of the components and their complex (Guinier and complex (Guinier and PP((rr) analyses, shape ) analyses, shape restoration)restoration)

Recall: Guinier AnalysisRecall: Guinier Analysis

Guinier showed that a plot (lnI(q) vs qGuinier showed that a plot (lnI(q) vs q2)2) gives a straight line of slope Rgives a straight line of slope Rgg

22/3 and I(0) /3 and I(0)

intercept that can be interpreted in terms intercept that can be interpreted in terms of the concentration, contrast and volume of the concentration, contrast and volume of the scattering particle. of the scattering particle.

3)0(ln)(ln

22 gR

qIqI

2)()0( VNI

Recall: Recall: II((qq) and ) and PP((rr) related by Fourier Transform) 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

SampleSample Protein Protein conc.conc.aa

(mg/ml)(mg/ml)RRgg (Å) (Å) DDmaxmax

(Å)(Å)

Porod Porod VolumeVolumebb (10(1033 x Å x Å33))

Calculated Calculated VolumeVolumecc

(10(1033 x Å x Å33))

MWMWdd

(kDa)(kDa)

SSRL dataSSRL data

NL1-638-NL1-638-(A&B)(A&B) 1.81.8 42.5 ± 0.442.5 ± 0.4 130130 209 ± 20209 ± 20 198198 130/151/144130/151/144

NL1/NX complexNL1/NX complex 3.63.6 46.8 ± 0.246.8 ± 0.2 155155 ** 275275 181/201/199181/201/199

University of Utah SAXSess instrument dataUniversity of Utah SAXSess instrument data

NL1-638-NL1-638-(A&B)(A&B) 13.213.2 38.3 ± 0.338.3 ± 0.3

7.67.6 40.1 ± 0.540.1 ± 0.5

4.14.1 41.4 ± 0.641.4 ± 0.6

Inf. dilutionInf. dilution 42.4 ± 0.642.4 ± 0.6 130130 208 ± 14 208 ± 14 198198 130/151/144130/151/144

NL1-638NL1-638 3.33.3 42.7 ± 0.742.7 ± 0.7 130130 250 ± 19250 ± 19 220220 136/166/160136/166/160

NL1-691NL1-691 3.83.8 51.8 ± 1.051.8 ± 1.0 165165 255 ± 26255 ± 26 257257 148/189/185148/189/185

NL2-615NL2-615 3.73.7 40.6 ± 0.640.6 ± 0.6 130130 178 ± 7178 ± 7 193193 135/146/140135/146/140

NL3-639NL3-639 1.21.2 40.3 ± 0.740.3 ± 0.7 130130 164 ± 12164 ± 12 190190 128/144/138128/144/138

NL4-619NL4-619 3.43.4 42.1 ± 0.642.1 ± 0.6 135135 199 ± 7199 ± 7 200200 132/140/145132/140/145

NL1/NX complexNL1/NX complex 19.719.7 40.9 ± 0.340.9 ± 0.3

15.715.7 40.8 ± 0.240.8 ± 0.2

9.89.8 44.0 ± 0.344.0 ± 0.3

8.78.7 43.7 ± 0.443.7 ± 0.4

6.66.6 44.5 ± 0.444.5 ± 0.4

4.54.5 45.2 ± 0.545.2 ± 0.5

3.83.8 47.7 ± 0.947.7 ± 0.9 155155 **

Inf. dilutionInf. dilution 47.7 ± 0.8 47.7 ± 0.8 155155 ** 275275 181/201/199181/201/199

NL1-638-Δ(A&B)

Complex

P(r

) arb

itra

ry u

nit

s

Distance (Angstroms)

0 10 20 30 40 50 60 70 80 90 100 110 120130 1401500

5

10

15

20

25

30

35

40

45

50

55265,851

228,139 ± 9,96547.1 ± 0.7Complex

199,26142.19 ± 0.7NL1-638-Δ(A&B)

Vol (Å3)Calculate

d

Vol (Å3)Experimenta

lRg (Å)Sample

184,172 ± 7,778

Determining the size of your Determining the size of your scattering particlescattering particle

Place data on an absolute scale (water Place data on an absolute scale (water scattering) and use:scattering) and use:

Orthaber et al. (2000) Orthaber et al. (2000) J. Appl. Cryst. 33J. Appl. Cryst. 33, 218, 218

Use a known mono-disperse protein Use a known mono-disperse protein scatterer (such as lysozyme) and use:scatterer (such as lysozyme) and use:

Krigbaum and Kugler (1970) Krigbaum and Kugler (1970) Biochemistry 9Biochemistry 9, 1216, 1216

Fischer et al. (2010) The molecular weight of proteins in solution can be Fischer et al. (2010) The molecular weight of proteins in solution can be determined from a single SAXS measurement on a relative scale. determined from a single SAXS measurement on a relative scale. J. Appl. J. Appl. Cryst. 43,Cryst. 43,101101

If you scale your data so that If you scale your data so that II(0) = 1, then: (0) = 1, then:

wherewhere

In practice we can only calculate:In practice we can only calculate:

So Fischer et al calculated correction factors to relate the ‘apparent’ So Fischer et al calculated correction factors to relate the ‘apparent’ volume using volume using Q’Q’ to the actual volume based on 1148 unique, known to the actual volume based on 1148 unique, known structures and their model profiles. structures and their model profiles.

V 22

Q

Q I(q)q2

0

dq

Q' I(q)q2qqmax

qmin

Planning the Planning the neutron scattering experimentneutron scattering experiment

Choose your data collection strategy Choose your data collection strategy (solvent matching or contrast variation?)(solvent matching or contrast variation?)

Determine how much sample is neededDetermine how much sample is needed Decide which subunit to labelDecide which subunit to label What deuteration level is needed in the What deuteration level is needed in the

labeling subunitlabeling subunit See MULCh*

http://www.mmb/usyd.edu.au/NCVWeb/

*MULCh, Whitten et al, accepted J. Appl. Cryst. 2007

MULChMULCh

MMododULULes for the analysis of neutron es for the analysis of neutron CContrast ontrast variation data variation data Contrast, Contrast, computes neutron contrasts of the computes neutron contrasts of the

components of a complexcomponents of a complex RRgg,, analyses the contrast dependence of the radius of analyses the contrast dependence of the radius of

gyration to yield information relating to the size and gyration to yield information relating to the size and disposition of the labelled and unlabeled components disposition of the labelled and unlabeled components in a complexin a complex

CompostCompost, , decomposes the contrast variation data into decomposes the contrast variation data into composite scattering functions containing information composite scattering functions containing information on the shape of the lab\led and unlabeled on the shape of the lab\led and unlabeled components and their dispositionscomponents and their dispositions

Solvent matchingSolvent matching

Best used when you are interested in the Best used when you are interested in the shape of one component in a complex, shape of one component in a complex, possibly how it changes upon ligand possibly how it changes upon ligand binding or complex formation. binding or complex formation.

Requires enough of the component to be Requires enough of the component to be solvent matched to complete a contrast solvent matched to complete a contrast variation series to determine required variation series to determine required %D%D22O (~4 x 200-300 O (~4 x 200-300 L, ~5 mg/ml).L, ~5 mg/ml).

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

Solvent Match Point DeterminationSolvent Match Point Determination

Apical view

Front view

Side view

90°

90°

Co-refinement of the Co-refinement of the neurexin positions and neurexin positions and

orientations with respect orientations with respect to NL1 give a model to NL1 give a model

against the X-ray and against the X-ray and neutron data gives us a neutron data gives us a model that we can map model that we can map

autism-linked mutations autism-linked mutations

R451CR451C

V403MV403M

K378RK378R

G99SG99S

Comoletti, Grishaev, Whitten et al. Structure 15, 693-705, 2007.

Superposition of solution scattering and Superposition of solution scattering and crystal structure for NL-NXcrystal structure for NL-NX

Contrast variationContrast variation To determine the shapes and To determine the shapes and

dispositions of labeled and unlabelled dispositions of labeled and unlabelled components in a complexcomponents in a complex

Requires Requires 5 x 200-300 5 x 200-300L (= 1 – L (= 1 – 1.5mL) of your labeled complex at 1.5mL) of your labeled complex at 5 5 mg/ml .mg/ml .

Deuteration level in labeled protein Deuteration level in labeled protein depends upon its size. depends upon its size. Smaller components require higher levels Smaller components require higher levels

of deuteration to be distinguished.of deuteration to be distinguished. Ideally would like to be able to take data Ideally would like to be able to take data

at the solvent match points for the labeled at the solvent match points for the labeled and unlabeled components and unlabeled components

Measure sample and solvent Measure sample and solvent blanks at each contrast point (use blanks at each contrast point (use a broad range of Da broad range of D22O O concentrations; e.g. 0,20,40, 80, concentrations; e.g. 0,20,40, 80, 100% D100% D22O)O)

Subtract solvent blank data from Subtract solvent blank data from samplesample

Sample to low-Sample to low-qq with sufficient with sufficient frequency to determine large frequency to determine large distances accurately (min. 15-20 distances accurately (min. 15-20 points in the Guinier region) points in the Guinier region)

Measure to high enough Measure to high enough qq to aid in to aid in checking background subtraction checking background subtraction ((qq = 0.45 = 0.45 ÅÅ-1-1))

qq = 0.01 -.45 is typical range for 10- = 0.01 -.45 is typical range for 10-150 kDa particles, usually requires 150 kDa particles, usually requires two detector positionstwo detector positions

Effects of incoherent scattering Effects of incoherent scattering from from 11H on backgroundsH on backgrounds

HCaM HCaM measurement was measurement was done in 42% Ddone in 42% D22O O to solvent match to solvent match the HCaM. the HCaM. Objective was to Objective was to see DCaM in see DCaM in presence of HCaM, presence of HCaM, but without but without interference from interference from HCaMHCaM

Incoherent Incoherent scattering from scattering from 11H H is a constant with qis a constant with q

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

Use Use RgRg (from MULCh) for Sturhman analysis (from MULCh) for Sturhman analysis

2222)( Dff DHDH

22222 )()( DffRRff HDDHDHDH

2222 DffRfRfR DHDDHHm

222

mobs RR

RKinA = 25.40 ÅRSda = 25.3 ÅD = 27.0 Å

Use Use CompostCompost (from (from MULCh) to solve for MULCh) to solve for

I(q)I(q)1111, , I(q)I(q)2222, , I(q)I(q)1212

II11 II1212

II22

)()()()( 1221222211

21 qIqIqIqI

Use SASREF7 to do rigid body Use SASREF7 to do rigid body refinement of the components refinement of the components against the scattering data (if you against the scattering data (if you

have pdb files for components)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

Incorporation of deuterium up to 86% of the chemically Non-exchangeable protons can be obtained by using D2O 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

Neutron scattering sample cellsNeutron scattering sample cells Helma quartz cells (high precision path-length, Helma quartz cells (high precision path-length,

suprasil) – need lots of them! suprasil) – need lots of them! Banjo-style (280 Banjo-style (280 L per 1 mm path length) or L per 1 mm path length) or

rectangular (170 rectangular (170 L per 1 mm path length) cells L per 1 mm path length) cells can be usedcan be used

Path lengths are only good to 1%, so good idea Path lengths are only good to 1%, so good idea to measure sample and solvent background in to measure sample and solvent background in the same cell if practical, but experiment the same cell if practical, but experiment logistics may prohibit that, so often have to logistics may prohibit that, so often have to ‘fudge’ background subtractions ‘fudge’ background subtractions

High incoherent scattering for High incoherent scattering for 11H means you H means you always want always want 1mm 1mm 11HH22O in the neutron beam O in the neutron beam to avoid multiple scattering to avoid multiple scattering

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/

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 1H 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 Assessing the quality of small-angle scattering resultssmall-angle scattering results

Are there instrumental effects unaccounted for?Are there instrumental effects unaccounted for? Are the scattering particles mono-disperse and identical or is there Are the scattering particles mono-disperse and identical or is there

a conformational ensemble?a conformational ensemble? Do you have dilute solution conditions?Do you have dilute solution conditions? Do the data show the expected Guinier and Porod behavior?Do the data show the expected Guinier and Porod behavior? Is the Is the PP((rr) “well-behaved?”) “well-behaved?” Are background subtractions accurate?Are background subtractions accurate? Have standards been measured?Have standards been measured? How well characterized is the sample (purity, concentration)How well characterized is the sample (purity, concentration) Are errors appropriately handled – can you rely on Are errors appropriately handled – can you rely on 22? ?

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