rigaku biosaxs webinar 093009

SAXS techniques f ifor proteins

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SAXS techniques for proteinsSAXS techniques for proteins

Bi SAXS li ti• BioSAXS applications• BioSAXS theoretical overview• Experimental hardware for the home lab• Application examplesApplication examples• References and resources

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SAXS techniques for proteins

Bi SAXS li ti


• BioSAXS applications• BioSAXS theoretical overview• Experimental hardware for the home lab• Application examplesApplication examples• References and resources

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Biological applications of SAXSBiological applications of SAXS

• Calculation of generalized structural parameters (Dmax, Rg)• Determination of the molecular shape of macro-moleculesp• Differentiation of mono-disperse and aggregated solutions• Differentiation of folded and unfolded protein solutions

Characterization of oligomeric states• Characterization of oligomeric states

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Bi SAXS li ti



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Crystallography vs SAXSCrystallography vs. SAXS

Integrated profile

Log(

I)

q [Å-1]

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Complimentary techniquesComplimentary techniques

Method Crystallography SAXSSamples Single crystals Dilute solutions (1 ~ 100mg/ml)

Advantages High resolution (up to 0.1nm)At i t t i f ti

Analysis in native conditionsAtomic structure information

Limitations Crystal required Low resolution (~1-2nm)Modeling ambiguity

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Scattering intensityScattering intensity)(*)(∝)( AAI

X-ray diffraction

)(*•)( ∝)( qAqAqI

xy

z∑

1=)}++(2exp{ =)(

N

jjjjjhkl lzkyhxiπfA q

iA d)()(Δ)( ∫

x

rSmall angle x-ray scattering

riqrrρqA d)exp()(Δ =)( ∫V

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Pair distribution functionPair distribution functionCrystal Solution

Patterson function Pair distribution function

P(r)

9r

Profile conversionProfile conversionSAXS pattern) SAXS pattern

Log(

I)q [Å-1]

Guinier plot

n(I)

Kratky plot

q2I

Pair distributionfunction

P(r

)q2 [Å-2]

ln

q [Å-1] r [Å]P

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q [Å ] q [Å ] r [Å]

Guinier plotGuinier plot32Rq

3)]0(ln[=)](ln[ GRqIqI

Mono-disperse Aggregated

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Kratky plotKratky plot

Folded Partially unfolded UnfoldedFolded Partially unfolded Unfolded

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Pair distribution functionPair distribution function∞

qqrqqIπr

rP d)sin()( 2

=)( ∫∞

02

DD maxmax

rrPrrPrR d)(d)( = ∫∫maxmax

0

2

0

2G

Dmax

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Shapes & scattering patternsShapes & scattering patternsSAXS patterns Pair distribution functionsSAXS patterns Pair distribution functions

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Refinement of molecular envelopeRefinement of molecular envelope

Squeeze a bean bag Compare Pcal(r) and Pobs(r)

Pcal(r)

Pobs(r)obs( )

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Pinhole SAXS cameraPinhole SAXS camera

XG

Optic

1st 3rd

Pinholes

2nd

Sample chamberBeam path

2D detector

3 m~ 3 m

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MicroMax 007MicroMax 007

MM007MM007

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MicroMax 002+MicroMax 002+

MM002+

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MicroMax 007 / 002+ specsMicroMax 007 / 002+ specsMM007 MM002+MM007 MM002+

Camera length ~ 3 m

S l i 1 5 5 15 lSample size 1.5 x 5 mm, 15 μl

Beam size at sample 0.5 mm

C K fl t l 1 108 2 107Cu Kα flux at sample 1 x 108 cps 2 x 107 cps

2θ minimum 0.1 º

Q minimum 0 006 Å-1Q minimum 0.006 Å 1

Maximum length scale 100 nm

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SMAX 3000SMAX 3000Dual Chamber SAXS Camera

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SMAX 3000SMAX 3000Simultaneous WAXS/SAXS

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Length scales for SAXS/WAXSLength scales for SAXS/WAXS

Sample-to-detector distance

qmin (nm-1) qmax (nm-1) Dmax (nm) Dmin (nm)

1500 mmMW-SAXS 0.0054 0.16 115 3.8

500500 mmMW-MAXS 0.16 4.8 3.8 1.2

30 mm 4 6 45 1 3 0 14IP-WAXS 4.6 45 1.3 0.14

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Flow cell sample handlingFlow cell sample handling

SamplePositioning stageCooling water

HeaterX-ray beam

Sample feeder

Linkam high temp unitManual flow cell

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Raw SAXS dataRaw SAXS dataHBsAg (hepatitis B surface antigen)

M t ditiMeasurement conditionsConcentration 5.75 mg/mlX-ray source MM007Scan time 60 min

C t f J h R U i it f G i

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Courtesy of John Rose, University of Georgia

Integrated SAXS dataIntegrated SAXS dataHBsAg (hepatitis B surface antigen)

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Integrated buffer dataIntegrated buffer dataHBsAg (hepatitis B surface antigen)

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Background corrected dataBackground corrected dataHBsAg (hepatitis B surface antigen)

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Guinier plot – determination or RGuinier plot determination or RgHBsAg (hepatitis B surface antigen)

Rg = 132. +/- 1.24

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Pair distribution functionPair distribution functionHBsAg (hepatitis B surface antigen)

R 132 04Rg = 132.04

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Ab-initio envelope determinationAb-initio envelope determinationHBsAg (hepatitis B surface antigen)

1 2 3

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Raw SAXS dataRaw SAXS dataHSA (human serum albumin)

M t ditiMeasurement conditionsConcentration 5 mg/mlX-ray source MM007Scan time 90 min

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Integrated SAXS dataIntegrated SAXS dataHSA (human serum albumin)

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Kratky plotKratky plotHSA (human serum albumin)

Well folded

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Pair distribution functionPair distribution functionHSA (human serum albumin)

R 29 02Rg = 29.02

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Crystal structure andymolecular envelope (HSA)

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Raw SAXS dataRaw SAXS dataG3B0F (C terminal of agrin)

M t ditiMeasurement conditionsConcentration 4 mg/mlX-ray source MM002+Scan time 120 min

Courtesy of Trushar Patel, University of Manitoba

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y , y

Integrated SAXS dataIntegrated SAXS dataG3B0F (C terminal of agrin)

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Pair distribution functionPair distribution functionG3B0F (C terminal of agrin)

Rg = 52.4

Characteristic shape of multi-domain protein

g 5Dmax = 175

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Rigid body refinementRigid body refinementG3B0F (C terminal of agrin)

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Comparison with synchrotron dataComparison with synchrotron data

Structure envelopes

Courtesy of Thomas Grant, HWI

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Biological Small AngleBiological Small Angle Scattering Group - DESY

• ATSAS 2.2• Software download

htt // bl h b d /E t lI f /http://www.embl-hamburg.de/ExternalInfo/Research/Sax/software.html

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Review papersReview papers• Robust high throughput solution structural analyses by small• Robust, high-throughput solution structural analyses by small

angle X-ray scattering (SAXS)Greg L Hura et al., Nature Methods, July 20 (2009)

• Small-angle scattering studies of biological macromolecules in solutionDmitri I Svergun et al Rep Prog Phys 66 1735 (2003)Dmitri I Svergun et al., Rep. Prog. Phys. 66 1735 (2003)

• X-ray solution scattering (SAXS) combined with crystallography and computationcrystallography and computationChristopher D Putnam et al., Q. Rev. Biophys. 40, 191 (2007)

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Reference bookReference book

S ll l tt i f X• Small-angle scattering of X-raysAndre Guinier & Gerard FournetWil (1955) ( t f i t)Wiley (1955) (out-of-print)

ProQuest www.umi.com

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ConferenceConferenceTh 67th A l Pitt b h Diff ti C f• The 67th Annual Pittsburgh Diffraction ConferenceOctober 29th – 31stUniversity of Georgia Center for Continuing Educationhttp://www.pittdifsoc.org/PDC_2009/

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Presentation for downloadPresentation for downloadThi t ti i il bl f d l d t• This presentation is available for download at http://www.rigaku.com/protein/webinars.html

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Thank you !a youw w w . R i g a k u . c o m

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rigaku biosaxs webinar 093009

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