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SAXS techniques for f proteins i

SAXS techniques for proteins


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

SAXS techniques for proteins


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

Biological applications of SAXS

Calculation of generalized structural parameters (Dmax, Rg) Determination of the molecular shape of macro-molecules p Differentiation of mono-disperse and aggregated solutions Differentiation of folded and unfolded protein solutions Characterization of oligomeric states

SAXS techniques for proteins


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

Crystallography vs SAXS vs.

Log(I)

Integrated profile

q [-1]

Complimentary techniques

Method Samples Advantages Limitations

Crystallography Single crystals High resolution (up to 0.1nm) Atomic t t At i structure information i f ti Crystal required

SAXS Dilute solutions (1 ~ 100mg/ml) Analysis in native conditions Low resolution (~1-2nm) Modeling ambiguity

Scattering intensity
I ( q) A( q) A * ( q)

X-ray diffraction
Ahkl (q ) = f j exp{2i ( hx j + ky j + lz j )}
j =1 N

z y x r

Small angle x-ray scattering


A( q) = ( r ) exp(i )dr (iqr
V

Pair distribution function


Crystal Solution

Patterson function

Pair distribution function

P(r)

r
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Profile conversion
Log(I) )

SAXS pattern

q [-1]

Guinier plot
ln n(I) q2I

Kratky plot
P P(r)

Pair distribution function

q2 [-2]

q [-1]

r []
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Guinier plot
3 q 2 RG ln[ I ( q)] = ln[ I (0)] 3

Mono-disperse

Aggregated

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Kratky plot

Folded

Partially unfolded

Unfolded

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Pair distribution function


r P( r ) = 2 2
Dmax

I (q)q sin(qr )dq


0 Dmax

RG 2 =

r P P ( r ) dr ( r ) dr
2 0 0

Dmax

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Shapes & scattering patterns


SAXS patterns Pair distribution functions

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Refinement of molecular envelope


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

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SAXS techniques for proteins


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

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Pinhole SAXS camera


Optic XG 1st 2nd 3rd Pinholes Sample chamber Beam path 2D detector

~3m

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MicroMax 007

MM007

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MicroMax 002+

MM002+

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MicroMax 007 / 002+ specs


MM007 Camera length Sample size S l i Beam size at sample Cu K flux t C K fl at sample l 2 minimum Q minimum Maximum length scale 1 x 108 cps 0.1 0.006 1 0 006 -1 100 nm ~3m 1.5 1 5 x 5 mm, 15 l l 0.5 mm 2 x 107 cps MM002+

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SMAX 3000 Dual Chamber SAXS Camera

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SMAX 3000 Simultaneous WAXS/SAXS

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Length scales for SAXS/WAXS


Sample-todetector distance 1500 mm MW-SAXS 500 mm MW-MAXS 30 mm IP-WAXS qmin (nm-1) qmax (nm-1) 0.0054 0.16 4.6 46 0.16 4.8 45 Dmax (nm) 115 3.8 1.3 13 Dmin (nm) 3.8 1.2 0.14 0 14

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Flow cell sample handling


Cooling water Positioning stage X-ray beam Sample Heater

Sample feeder

Manual flow cell

Linkam high temp unit

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SAXS techniques for proteins


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

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Raw SAXS data


HBsAg (hepatitis B surface antigen)
Measurement conditions M t diti Concentration X-ray source Scan time 5.75 mg/ml MM007 60 min

Courtesy of John Rose, U i C t fJ h R University of G it f Georgia i


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Integrated SAXS data


HBsAg (hepatitis B surface antigen)

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Integrated buffer data


HBsAg (hepatitis B surface antigen)

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Background corrected data


HBsAg (hepatitis B surface antigen)

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Guinier plot determination or Rg


HBsAg (hepatitis B surface antigen)

Rg = 132. +/- 1.24

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Pair distribution function


HBsAg (hepatitis B surface antigen)

Rg = 132 04 132.04

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Ab-initio envelope determination


HBsAg (hepatitis B surface antigen)

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Raw SAXS data


HSA (human serum albumin)
Measurement conditions M t diti Concentration X-ray source Scan time 5 mg/ml MM007 90 min

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Integrated SAXS data


HSA (human serum albumin)

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Kratky plot
HSA (human serum albumin) Well folded

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Pair distribution function


HSA (human serum albumin)

Rg = 29 02 29.02

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Crystal structure and y molecular envelope (HSA)

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Raw SAXS data


G3B0F (C terminal of agrin)
Measurement conditions M t diti Concentration X-ray source Scan time 4 mg/ml MM002+ 120 min

Courtesy of Trushar Patel, University of Manitoba y , y


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Integrated SAXS data


G3B0F (C terminal of agrin)

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Pair distribution function


G3B0F (C terminal of agrin) Characteristic shape of multi-domain protein
Rg = 52.4 5 Dmax = 175

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Rigid body refinement


G3B0F (C terminal of agrin)

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Comparison with synchrotron data


Structure envelopes

Courtesy of Thomas Grant, HWI

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SAXS techniques for proteins


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

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Biological Small Angle Scattering Group - DESY


ATSAS 2.2 Software download http://www.embl-hamburg.de/ExternalInfo/ htt // bl h b d /E t lI f / Research/Sax/software.html

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Review papers
Robust, high throughput 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 solution Dmitri I Svergun et al Rep Prog Phys 66 1735 (2003) al., Rep. Prog. Phys. X-ray solution scattering (SAXS) combined with crystallography and computation Christopher D Putnam et al., Q. Rev. Biophys. 40, 191 (2007)

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Reference book
S ll Small-angle scattering of X-rays l tt i fX Andre Guinier & Gerard Fournet Wiley Wil (1955) ( t f i t) (out-of-print) ProQuest www.umi.com

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Conference
Th 67th Annual Pittsburgh Diffraction Conference The A l Pitt b h Diff ti C f October 29th 31st University of Georgia Center for Continuing Education http://www.pittdifsoc.org/PDC_2009/

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Presentation for download


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

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Thank you ! a
www.Rigaku.com

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