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Exploring Protein and Nucleic Acid Structure with Small Angle
X-ray Scattering (SAXS)
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Welcome
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Samuel Butcher, Ph.D.Professor, BiochemistryUniversity of Wisconsin - [email protected]+1.608.2263.3890
Matt Benning, Ph.D.Sr. Applications Scientist, SC-XRDBruker AXS [email protected]+1.608.276.3819
Brian Jones, Ph.D.Sr. Applications Scientist, XRDBruker AXS [email protected]+1.608.276.3088
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Overview
IntroductionNanoSTAR HardwareBioSAXS ExperimentApplications
Radius of gyrationMolecular weightFolding / unfoldingPair distribution functionsShape reconstructionStructure determination
Combined NMR-SAXS ApproachQ & A
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Introduction
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The SAXS Experiment
d
Incident x-ray beam
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The SAXS Experiment
d
nλ = 2dsinθ
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XRDDiffraction at crystal latticeDiffraction angles: 4 - 170°
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The SAXS Experiment
d
SAXSScattering at particles
Scattering angles: 0 - 4°
XRDDiffraction at crystal latticeDiffraction angles: 4 - 170°
nλ = 2dsinθ
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The SAXS Experiment
d
SAXSScattering at particles
Scattering angles: 0 - 4°
d 1 – 100nm
XRDDiffraction at crystal latticeDiffraction angles: 4 - 170°
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nλ = 2dsinθ
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Scattering vector q
λθπ /sin4≡q
2θ
ki
ks
q
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Amplitude of scattering
Difference in scattering density between volume element at r with macromolecule and that of solvent.
Scattering intensity given by:
SAXS scattering intensity
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( ) rderqAV
rqis
rr rr
∫ −= ρρ )()(
)(*)()( qAqAqI =
sr ρρ −)(r
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Example SAXS scattering
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Example SAXS scattering
2θ = 0°
2θ = 4°
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Example SAXS scattering
Azimuthially Averaged SAXS Scattering
q [Å-1]
0.01 0.1
Inte
nsity
[a.u
.]
1
10
100
1000
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Small angle X-ray scattering (SAXS)
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NanoSTAR Hardware
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NanoSTAR
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Experimental setup
X-ray source
Multilayer optics Pinhole system Sample Evacuatedbeam path
Beam stop
Detector
XY-Stage Reference sample wheel
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SourceIμS - Incoatec Microfocus Source
High intensity at only 30 W
No water cooling required
Long lifetime without maintenance3 year warranty
Low cost of ownership
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SourceTurbo X-ray Source (TXS)
Highest intensity X‐ray sourceDirect drive anode allows efficient cooling higher power
Small filament size higher power density
Alignment-free filament mounting
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Next Generation SourceCENTAURUS - Metal-Jet
Ga (95%)/In/Sn alloy liquid metal jet, 200 µm wide, 50 m/s velocity
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Exclusive collaboration with Excillum – spinoff from KTH, Stockholm, Sweden
Spot size: 5 – 20 μm
Emission: Ga Kα, 9.25 keV
Power loading: >500 kW/mm2
Brightness: > 10x rotating anode
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Next Generation SourceCENTAURUS - Metal-Jet
Main components based on proven technology
Electron gun and focusing
Standard HV supply and existing HV insulators used
Cathode and optics “borrowed” from accelerator technology
Liquid jet target
Standard, reliable industrial process pump used
Nozzle design “borrowed” from water jet cutting technology
Closed loop recycling system, no dynamic vacuum seals
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Arrangement two identical mirrors in a side-by-side configuration
Benefits:more compacteasy alignmentsymmetrical divergence spectrum
OpticsMontel-P Multilayer Mirror
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Collimation3 Pinhole Source to Sample
Beam size at sample: 150 μm or 400 μm diameterRequires small amounts of sample
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Sample HolderBioSAXS cells
Reusable, vacuum sealed quartz capillary tube cells
Software controlled X-Y drive can be used as an autochanger to bring each to the beam automatically
Y drive
X drive
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DetectorVÅNTEC-2000 2D Mikro-GapTM
High sensitivityReal-time photon counter
Extremely low background<0.0005 cps/mm2
Good spatial resolution70 – 100 μm pixel size
Large active areaEntire scattering range in 1 exposure
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Variable Sample to Detector Distance
SAXS1070 mm670 mm
WAXS270 mm130 mm60 mm
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Accessible scattering range for different sample to detector distances
Distance (mm) q min (Å‐1)d max (Å)
q max (Å‐1)d min (Å)
1070 0.005
1250
0.28
24
670 0.01
600
0.4
16
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Full 2D Detector Integration Advantage
q [Å-1]
0.01 0.1
Inte
nsity
[a.u
.]
10
100
Full 2D Integration
Partial 2D Integration
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BioSAXS Experiment
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BioSAXS Sample
Samples typically consist of biological macromolecules and their complexes (proteins, DNA and RNA) in solution
Homogeneous
Monodisperse
Concentration: >1 mg/ml
Sample amount: 10-15 μl
Perfectly matched buffer solution provided for solvent blank measurement
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NanoSTAR BioSAXS Cells
Reusable, vacuum-tight, quartz capillaries
Used as a flow-through cell
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Loading Samples
Deposit into cellDraw up into syringe/pipette
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Loading Samples
LoadSeal
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Sample alignment
Center of cell is aligned to the x-ray beam by automatically scanning in x and y direction using nanography.
Y - direction
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BioSAXS sample requirements
Data collection and data treatment via windows GUI
1D intensity vs scattering vector (q) exported
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BioSAXS analysis softwareEMBL - European Molecular Biology Lab
ATSAS – program suite for small angle scattering data analysis from biological macromolecules.
Data from NanoSTAR can be directly imported.
http://www.embl-hamburg.de/
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Applications
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SAXS information
The radius of gyration (Rg), a parameter characterizing shape and size, can be quickly estimated from the low angle scattering
Determining the scattering intensity at 2θ=0, I(0), can be used to estimate molecular weight
A Fourier transform of the scattering curve results in the pair distance distribution function giving a more precise value of Rg and I(0), the maximum linear dimension of the protein, Dmax
The distribution function can be used as input for ab initio structure determination algorithms which produce three-dimensional models called “envelopes” that describe size and shape
The distribution function can be combined with other high-resolution techniques, such as NMR or X-ray crystallography, to create a complete and accurate image of the entire macromolecule
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Guinier plot
The slope yields the radius of gyration, Rg Extrapolation to q=0 gives I(0)
Rgq < 1.3
Guinier approximation: )
31( 22
)0()( gRqeIqI
−=
3)]0(ln[)](ln[
22gRq
IqI −=
Adapted from Tainer et al, Quarterly Reviews of biophysics 2007
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Molecular weight determination
The molecular weight of a sample can be determined using I(0)
Adapted from Tainer et al, Quarterly Reviews of biophysics 2007
Calibrated with a standard protein
Accurate Protein concentration
Partial specific volume is assumed to be the same
Error ~ 10 %, Svergun 2007
dards
dardssamplesample I
MWIMWtan
tan
)0()0( ×=
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Kratky plot
The Kratky plot is a useful tool for studying dynamics
Globular proteins follow Porod’s law and have bell-shaped curves
Extended molecules lack this peak and plateau in the larger q-range
Adapted from Tainer et al, Quarterly Reviews of biophysics 2007
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Pair-distance distribution function
Provides information about the distances between scatters
Corresponds to the Patterson function in crystallography
Adapted from Tainer et al, Quarterly Reviews of biophysics 2007
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Pair-distance distribution function
Alternative method for calculation Rg and I(0)Assignment of Dmax, max. linear dimension of a scattering particleP(r) can be calculated from atomic models
dqqR
qRqIRP )sin()(21)(
02 ∫∞
=π
Dmax
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Adapted from Svergun and Koch, Rep. Prog. Phys. 2003
Pair-distance distribution function
Can provide useful information about the shape of the molecule
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Urate Oxidase
URATE OXIDASE from Aspergillus flavus provided by the Protein Data Bank (PDB 1R56)
Bruker NanoSTAR
Concentration: 17 mg/ml
The red lines give the fit of the Fourier Transform of the pair-distance distribution function p(r) to the experimental data
Rg = 31.28 ± 0.03 Å R= 40.4 ÅI(0) = 1.230 ± 0.003 cm-1Dmax = 82 Å
q [Å-1]
0.0 0.1 0.2 0.3
dσ/dΩ
[cm
-1]
0.001
0.01
0.1
1
10
ExperimentFit
Pair Distance Distribution Function
r [Å]
0 20 40 60 80
p(r)
0.0000
0.0005
0.0010
0.0015
0.0020
0.002517.0 mg/mL
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Ab initio protein shape reconstruction
Generation of a low resolution 3-D envelope from 1-D scattering pattern
The most commonly used approach is approximating the electron density in terms of an assembly of beads or dummy atoms
Employs a Monte Carlo-based algorithm to find the model that fits the scattering data
Impose constraints to produce a more realistic model (compact and connected)
The final result may not be unique, several models may provide a equally good fit to the data
Compare and average results from different reconstruction runs
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Estrogen receptor alpha activation by calmodulin
Dr. Jeff Urbauer, University of Georgia
Studies have implied a role for Ca2+ and Calmodulin in breast carcinoma
Define structural changes in ERα that accompany calmodulin binding
Combination of NMR and SAXS techniques since complex is difficult to crystallize
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Construct of the ligand binding domain complexed with CaM
From chemical shift data it appears that the bound CaM is in a more extended state
As a control, collect data on CaM complexed with a peptide corresponding to the CaM binding region of smooth muscle MLCK which forms a very compact structure
Experimental
Bruker NanoSTAR
Exposure time: 60 minutes
Sample concentration: 10 mg/ml
Estrogen receptor alpha activation by calmodulin
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1-D scattering curve buffer correctedusing PRIMUS
PRIMUS: J.Appl. Cryst. 36, 1277-1282
Calmodulin with MLCK peptide Calmodulin with ERα domain
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Guinier analysis
Calmodulin with MLCK peptide Calmodulin with ERα domain
Rg = 17.51 Å Rg = 18.53 Å
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Pair distance distribution functionsusing GNOM
GNOM: Svergun, D.I. (1992) J.Appl. Cryst. 25, 495-503
Rg = 17.08 Å
Dmax = 52 Å
Rg = 19.65 Å
Dmax = 63 Å
Calmodulin with MLCK peptide Calmodulin with ERα domain
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Shape reconstruction
The envelope calculated from the SAXS data for the CaM-MLCK peptide superimposes very well with the high resolution structure determined by NMR
The Rg calculated from the SAXS data was identical to that determined from the NMR structure
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Ab initio structure from DAMMIF*
*Franke, D. and Svergun, D.I. (2009). J. Appl. Cryst., 42, 342-346
Calmodulin with MLCK peptide Calmodulin with ERα domain
Calmodulin – ERα envelope suggests a more extended confirmation for the complex which is consistent with chemical shift data
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References
X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution
John Tainer et al., Q. Rev. Biophys. 40, 191 (2007)
Small-angle scattering studies of biological macromolecules in solution
Dmitri I Svergun et al., Rep. Prog. Phys. 66 1735 (2003)
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Biomolecular structure using a combined SAXS and NMR approach
Sam ButcherDept. of Biochemistry, UW-Madison
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NMR size limitation is 30-40 kDa for RNAs and RNPs
38.5 kDa RNA‐protein complexZhang et al. (Summers) 2007
30 kDa RNA‐Mn++ complexDavis et al. 2007
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Recent examples of the combined NMR-SAXS approach
Grishaev, Wu, Trewhella & Bax, 2005. Refinement of multidomain protein structures by combination of solution small angle x-ray scattering and NMR data. J. Am. Chem. Soc. 127, 16621-16628
Grishaev et al., 2008. Solution structure of tRNAVal from refinement of homology model against residual dipolar coupling and SAXS data. J. Biomol. NMR 42(2):99-109.
Wang et al., 2009. Determination of multicomponent protein structures in solution using global orientation and shape restraints. J. Am. Chem. Soc. 131(30):10507-15.
Zuo et al., 2009. Global molecular structure and interfaces: refining an RNA:RNA complex structure using solution X-ray scattering data. J. Am. Chem. Soc. 130(11):3292-3.
Wu et al., 2009. A method for helical RNA global structure determination in solution using small-angle X-ray scattering and NMR measurements. J. Mol. Biol. 393, 717-734
Zuo et al., 2010. Solution structure of the cap-independent translational enhancer and ribosome-binding element in the 3' UTR of turnip crinkle virus. Proc. Natl. Acad. Sci.107(4):1385-90
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APS at ArgonneAdvanced Photon Source Synchrotron at Argonne, Ill.
Beamline 12-IDJordan Halsig58
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The Facility
Example of a benchtop SAXS instrument 900 MHz NMRNMRFAM UW-Madison
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An important RNA structure and application for SAXS: the GAAA tetraloop receptor
Cate, J. H., et al. (1996). Science 273:1678-1685 Adams, P.L., et al (2004). Nature 430: 45-50
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Rational design of an RNA homodimer
Jaeger , L., et al. (2000). Angew Chem Int Ed Engl 39 (14): 2521-2524
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NMR structure of the tetraloop receptor complex
• Davis et al., 2005. RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloop-receptor complex. J. Mol. Biol. 351(2):371-82
• Davis et al., 2007. Role of metal ions in the tetraloop-receptor complex as analyzed by NMR.J. Mol. Biol. 351(2):371-82
• NOE distance restraints: 700 x 2 • Intermolecular NOEs: 36 x 2 • Residual dipolar couplings (RDCs): 11 x 2• RMSD 1.0 Å
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Can SAXS data substitute for intermolecularNOE and H-bond restraints?
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Can SAXS data substitute for intermolecularNOE and H-bond restraints?
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SAXS-defined rigid body calculation with no intermolecular NOEs or H-bonds
r.m.s.d.= 0.4 ÅZuo et al., 2008 J. Am. Chem. Soc. 130, 3292-3293
vs. NMR structure
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Conclusion: SAXS data can be used to define molecular interfaces and can compensate for sparse NMR data
Hypothesis: Combination of SAXS + NMR data should lead to even more accurate structures
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NMR only vs. NMR + SAXS
r.m.s.d. = 3.2 ÅZuo et al., 2008 JACS 130, 3292-3293
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Rg
NMR 25.1
NMR+SAXS 23.1
Measured 23.0
(Å)
NMR + SAXS data leads to more accurate structures!
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Do we need a synchrotron?
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0.01 0.1
I (a
.u.)
0.1
1
10
100
1000
Bruker-AXS NanostarAPS Synchrotron beamline 12-ID
q [Å-1]
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Rg from synchrotron and Bruker NanoSTAR agree
Source Synchrotron Bruker NanoSTAR
Location APS beamline 12-ID
Bruker AXS Madison, WI
Radius of gyration 23.2 +/- 0.3 Å 23.3+/- 0.8 Å
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0.01 0.1
I
0.1
1
10
100
1000
q [Å‐1]
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Low resolution molecular envelope from benchtop SAXS data
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SAXS Workflow
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Dammin ab initio Shape Calculations
Dummy atom simulation
Back calculates scattering curve
Stops when error is acceptable
Svergun, D.I. 1999 Biophys J.
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Averaging with Damaver
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Volume model of U2/U6 RNA (111 nt)
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Possible configuration of helices
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Acknowledgements
Jordan Halsig (UW Madison)NMRFAM (UW Madison)John Markley (UW Madison)
Brian Jones (Bruker AXS)
Yun-Xing Wang (NCI)Xiaobing Zuo (NCI)Jinbu Wang (NCI)
Alex Grishaev (NIH)Ad Bax (NIH)
Jill Trewhella (U. Utah)Marc Taraban (U. Utah)
Supported by the National Science Foundation and the National Institutes of Health
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Q & APlease type any questions you may have in
the Q&A panel and then click Send.
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