exploring protein and nucleic acid structure with small …€¦ · · 2011-06-08exploring...

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

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°

The SAXS Experiment

d

SAXSScattering at particles

Scattering angles: 0 - 4°


nλ = 2dsinθ

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The SAXS Experiment

d

SAXSScattering at particles

Scattering angles: 0 - 4°

d 1 – 100nm


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nλ = 2dsinθ

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

Example SAXS scattering

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2θ = 0°

2θ = 4°

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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)

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

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

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

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670 0.01

600

0.4

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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

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

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

Molecular weight determination

The molecular weight of a sample can be determined using I(0)


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( ×=

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


Pair-distance distribution function

Provides information about the distances between scatters

Corresponds to the Patterson function in crystallography



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

Adapted from Svergun and Koch, Rep. Prog. Phys. 2003


Can provide useful information about the shape of the molecule

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

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

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

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

1-D scattering curve buffer correctedusing PRIMUS

PRIMUS: J.Appl. Cryst. 36, 1277-1282

Calmodulin with MLCK peptide Calmodulin with ERα domain

Guinier analysis


Rg = 17.51 Å Rg = 18.53 Å

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


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

Ab initio structure from DAMMIF*

*Franke, D. and Svergun, D.I. (2009). J. Appl. Cryst., 42, 342-346


Calmodulin – ERα envelope suggests a more extended confirmation for the complex which is consistent with chemical shift data

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)

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

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]

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]

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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exploring protein and nucleic acid structure with small …€¦ · · 2011-06-08exploring...

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