03 - Bruker - SAXS - Exploring Protein and Nucleic Acid Structure with Small Angle X-ray Scattering
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Exploring Protein and Nucleic Acid Structure with Small Angle X-ray Scattering (SAXS) 1 Welcome 2 Brian Jones, Ph.D. Matt Benning, Ph.D. Samuel Butcher, Ph.D. Sr. Applications Scientist, XRD Bruker AXS Inc. brian.jones@bruker-axs.com +1.608.276.3088 Sr. Applications Scientist, SC-XRD Bruker AXS Inc. matt.benning@bruker-axs.com +1.608.276.3819 Professor, Biochemistry University of Wisconsin - Madison butcher@biochem.wisc.edu +1.608.2263.3890 Overview Introduction NanoSTAR Hardware BioSAXS Experiment Applications 3 Radius of gyration Molecular weight Folding / unfolding Pair distribution functions Shape reconstruction Structure determination Combined NMR-SAXS Approach Q&A Introduction 4 The SAXS Experiment d Incident x-ray beam 5 The SAXS Experiment XRD nλ = 2dsinθ Diffraction at crystal lattice Diffraction angles: 4 - 170° d 6 The SAXS Experiment XRD nλ = 2dsinθ Diffraction at crystal lattice Diffraction angles: 4 - 170° d SAXS Scattering at particles Scattering angles: 0 - 4° 7 The SAXS Experiment XRD nλ = 2dsinθ Diffraction at crystal lattice Diffraction angles: 4 - 170° d SAXS Scattering at particles Scattering angles: 0 - 4° d Æ 1 – 100nm 8 Scattering vector q ks q 2θ ki q ≡ 4π sin θ / λ 9 SAXS scattering intensity Amplitude of scattering A(q ) = ∫ V rr r r iq r (ρ (r ) − ρ s )e dr Difference in scattering density between volume element at r with macromolecule and that of solvent. r ρ (r ) − ρ s Scattering intensity given by: I (q ) = A(q ) A * (q ) 10 Example SAXS scattering 11 Example SAXS scattering 2θ = 4° 2θ = 0° 12 Example SAXS scattering Azimuthially Averaged SAXS Scattering Intensity [a.u.] 1000 100 10 1 0.01 0.1 q [Å-1] 13 Small angle X-ray scattering (SAXS) 14 NanoSTAR Hardware 15 NanoSTAR 16 Experimental setup X-ray source Multilayer optics 17 XY-Stage Pinhole system Reference sample wheel Sample Beam stop Evacuated beam path Detector Source IμS - Incoatec Microfocus Source High intensity at only 30 W No water cooling required Long lifetime without maintenance 3 year warranty 18 Low cost of ownership Source Turbo X-ray Source (TXS) Highest intensity X‐ray source 19 Direct drive anode allows efficient cooling Æ higher power Small filament size Æ higher power density Alignment-free filament mounting Next Generation Source CENTAURUS - Metal-Jet Ga (95%)/In/Sn alloy liquid metal jet, 200 µm wide, 50 m/s velocity 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 20 Next Generation Source CENTAURUS - Metal-Jet Main components based on proven technology Electron gun and focusing Cathode and optics “borrowed” from accelerator technology Liquid jet target 21 Standard HV supply and existing HV insulators used Standard, reliable industrial process pump used Nozzle design “borrowed” from water jet cutting technology Closed loop recycling system, no dynamic vacuum seals Optics Montel-P Multilayer Mirror Arrangement Benefits: 22 two identical mirrors in a side-by-side configuration more compact easy alignment symmetrical divergence spectrum Collimation 3 Pinhole Source to Sample 23 Beam size at sample: 150 μm or 400 μm diameter Requires small amounts of sample Sample Holder BioSAXS cells Y drive X drive 24 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 Detector VÅNTEC-2000 2D Mikro-GapTM 25 High sensitivity Real-time photon counter Extremely low background <0.0005 cps/mm2 Good spatial resolution 70 – 100 μm pixel size Large active area Entire scattering range in 1 exposure Variable Sample to Detector Distance 26 SAXS 1070 mm 670 mm WAXS 270 mm 130 mm 60 mm Accessible scattering range for different sample to detector distances q min (Å‐1) q max (Å‐1) d max (Å) d min (Å) 1070 0.005 1250 0.28 24 670 0.01 600 0.4 16 Distance (mm) 27 Full 2D Detector Integration Advantage Full 2D Integration Intensity [a.u.] 100 10 Partial 2D Integration 0.01 0.1 q [Å-1] 28 BioSAXS Experiment 29 BioSAXS Sample Samples typically consist of biological macromolecules and their complexes (proteins, DNA and RNA) in solution 30 Homogeneous Monodisperse Concentration: >1 mg/ml Sample amount: 10-15 μl Perfectly matched buffer solution provided for solvent blank measurement NanoSTAR BioSAXS Cells 31 Reusable, vacuum-tight, quartz capillaries Used as a flow-through cell Loading Samples 32 Draw up into syringe/pipette Deposit into cell Loading Samples 33 Seal Load Sample alignment Y - direction 34 Center of cell is aligned to the x-ray beam by automatically scanning in x and y direction using nanography. BioSAXS sample requirements 35 Data collection and data treatment via windows GUI 1D intensity vs scattering vector (q) exported BioSAXS analysis software EMBL - European Molecular Biology Lab http://www.embl-hamburg.de/ 36 ATSAS – program suite for small angle scattering data analysis from biological macromolecules. Data from NanoSTAR can be directly imported. 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 Guinier approximation: I ( q ) = I ( 0) e 1 ( − q 2 R g2 ) 3 Rgq < 1.3 ln[ I ( q )] = ln[ I (0)] − q 2 Rg2 3 The slope yields the radius of gyration, Rg Extrapolation to q=0 gives I(0) 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 MW sample = I (0) sample × MW s tan dard I (0) s tan dard Accurate Protein concentration Partial specific volume is assumed to be the same Error ~ 10 %, Svergun 2007 Adapted from Tainer et al, Quarterly Reviews of biophysics 2007 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 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 Pair-distance distribution function Alternative method for calculation Rg and I(0) Assignment of Dmax, max. linear dimension of a scattering particle P(r) can be calculated from atomic models P( R) = 1 2π 2 ∞ ∫ I (q ) 0 sin(qR) dq qR Dmax Pair-distance distribution function Can provide useful information about the shape of the molecule Adapted from Svergun and Koch, Rep. Prog. Phys. 2003 Urate Oxidase URATE OXIDASE from Aspergillus flavus provided by the Protein Data Bank (PDB 1R56) 10 Experiment Fit -1 dσ/dΩ [cm ] 1 0.1 Pair Distance Distribution Function 0.0025 0.01 17.0 mg/mL 0.0020 0.001 0.1 0.2 0.3 q [Å-1] Bruker NanoSTAR Concentration: 17 mg/ml 0.0015 p(r) 0.0 0.0010 0.0005 0.0000 0 20 40 60 80 r [Å] 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 Å I(0) = 1.230 ± 0.003 cm-1 Dmax = 82 Å 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 Define structural changes in ERα that accompany calmodulin binding Studies have implied a role for Ca2+ and Calmodulin in breast carcinoma Combination of NMR and SAXS techniques since complex is difficult to crystallize Estrogen receptor alpha activation by calmodulin 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 1-D scattering curve buffer corrected using PRIMUS Calmodulin with MLCK peptide PRIMUS: J.Appl. Cryst. 36, 1277-1282 Calmodulin with ERα domain Guinier analysis Calmodulin with MLCK peptide Rg = 17.51 Å Calmodulin with ERα domain Rg = 18.53 Å Pair distance distribution functions using GNOM Calmodulin with MLCK peptide Rg = 17.08 Å Dmax = 52 Å GNOM: Svergun, D.I. (1992) J.Appl. Cryst. 25, 495-503 Calmodulin with ERα domain 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* 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 *Franke, D. and Svergun, D.I. (2009). J. Appl. Cryst., 42, 342-346 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 Butcher Dept. of Biochemistry, UW-Madison 55 NMR size limitation is 30-40 kDa for RNAs and RNPs 56 30 kDa RNA‐Mn++ complex Davis et al. 2007 38.5 kDa RNA‐protein complex Zhang et al. (Summers) 2007 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 57 Advanced Photon Source Synchrotron at Argonne, Ill. APS at Argonne Beamline 12-ID 58 Jordan Halsig The Facility Example of a benchtop SAXS instrument 59 900 MHz NMR NMRFAM UW-Madison 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 60 Rational design of an RNA homodimer Jaeger , L., et al. (2000). Angew Chem Int Ed Engl 39 (14): 2521-2524 61 NMR structure of the tetraloop receptor complex • NOE distance restraints: 700 x 2 • Intermolecular NOEs: 36 x 2 • Residual dipolar couplings (RDCs): 11 x 2 • RMSD 1.0 Å • Davis et al., 2005. RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloopreceptor 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 62 Can SAXS data substitute for intermolecular NOE and H-bond restraints? 63 Can SAXS data substitute for intermolecular NOE and H-bond restraints? 64 SAXS-defined rigid body calculation with no intermolecular NOEs or H-bonds vs. NMR structure r.m.s.d.= 0.4 Å Zuo et al., 2008 J. Am. Chem. Soc. 130, 3292-3293 65 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 66 NMR only vs. NMR + SAXS r.m.s.d. = 3.2 Å Zuo et al., 2008 JACS 130, 3292-3293 67 NMR + SAXS data leads to more accurate structures! Rg (Å) 68 NMR 25.1 NMR+SAXS 23.1 Measured 23.0 Do we need a synchrotron? 1000 I (a.u.) 100 10 1 0.1 0.01 q [Å-1] 0.1 Bruker-AXS Nanostar APS Synchrotron beamline 12-ID 69 Rg from synchrotron and Bruker NanoSTAR agree 1000 I 100 10 1 0.1 0.01 70 q [Å‐1] 0.1 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 Low resolution molecular envelope from benchtop SAXS data 71 SAXS Workflow 72 Dammin ab initio Shape Calculations Dummy atom simulation Back calculates scattering curve Stops when error is acceptable Svergun, D.I. 1999 Biophys J. 73 Averaging with Damaver 74 Volume model of U2/U6 RNA (111 nt) 75 Possible configuration of helices 76 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 77 Q&A Please type any questions you may have in the Q&A panel and then click Send. 78 Thank you for attending! A copy of the slides and a link to the recording will be emailed to you. Please take a moment to complete the brief survey on your screen. 79
