Nucleic Acid Structure Characterization by Small Angle X‐Ray Scattering (SAXS)

Jordan E. Burke1, Samuel E. Butcher1

1 Department of Biochemistry, University of Wisconsin, Madison, Wisconsin
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.18
DOI:  10.1002/0471142700.nc0718s51
Online Posting Date:  December, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Small angle X‐ray scattering (SAXS) is a powerful method for investigating macromolecular structure in solution. SAXS data provide information about the size and shape of a molecule with a resolution of ∼2 to 3 nm. SAXS is particularly useful for the investigation of nucleic acids, which scatter X‐rays strongly due to the electron‐rich phosphate backbone. Therefore, SAXS has become an increasingly popular method for modeling nucleic acid structures, an endeavor made tractable by the highly regular helical nature of nucleic acid secondary structures. Recently, SAXS was used in combination with NMR to filter and refine all‐atom models of a U2/U6 small nuclear RNA complex. In this unit, general protocols for sample preparation, data acquisition, and data analysis and processing are given. Additionally, examples of correctly and incorrectly processed SAXS data and expected results are przovided. Curr. Protoc. Nucleic Acid Chem. 51:7.18.1‐7.18.18. © 2012 by John Wiley & Sons, Inc.

Keywords: nucleic acid structure; SAXS; NMR; RNA folding; molecular modeling

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Sample Preparation
  • Basic Protocol 2: SAXS Data Acquisition
  • Basic Protocol 3: SAXS Data Processing and Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Sample Preparation

  Materials
  • Nucleic acid sample
  • SAXS buffer
  • Tris base, 99% pure
  • 0.3 M sodium acetate, pH 5.2
  • Ethanol
  • Potassium hydroxide (KOH), 89% pure
  • Liquid nitrogen
  • 15‐mL Centrifuge tubes (Falcon)
  • 90°C heating block
  • 1‐L, 0.22‐µm filter flask (Millipore or GE Healthcare)
  • 0.5‐ and 15‐mL centrifugal filters, regenerated cellulose (Amicon, Ultra)
  • Centrifuge
  • HiLoad 16/60 Superdex 75 or 200 prep‐grade column (GE Healthcare)
  • FPLC system (such as ÄKTAFPLC) equipped with online UV monitor at 254 nm, 100‐µL loading loop, and fraction collector
  • 100‐µL syringe with 23‐mm, 22‐G, blunt‐end needle (Hamilton)
  • Nanodrop ND‐1000 spectrophotometer or other UV spectrophotometer
  • 0.5‐ to 3‐mL dialysis cassettes (Slide‐A‐Lyzer, Thermo Scientific)
  • 0.20‐µm syringe filters (Sartorius Stedim)
  • 0.5‐ or 1.5‐mL microcentrifuge tubes (Eppendorf)
  • 0.22‐µm SpinX cellulose acetate filters (CoStar)
  • Microcentrifuge
  • Additional reagents and equipment for non‐denaturing polyacrylamide gel electrophoresis (PAGE; unit 10.4)

Basic Protocol 2: SAXS Data Acquisition

  Materials
  • Silver(I) behenate (AgBe)
  • Reserved filtered dialysate (see protocol 1)
  • Purified nucleic acid (see protocol 1)
  • Standard sample: lysozyme (Fluka), glucose isomerase (Hampton research), or standard RNA sample (Table 7.18.1)
  • SAXS instrument (∼67 to 100 cm sample‐to‐detector distance with 1‐ to 1.5‐mm quartz capillary and glassy carbon)
  • Additional detector for WAXS (30 cm sample‐to‐detector distance), optional
    Table 7.8.1   MaterialsSAXS Standard Samples

    Molecule (conditions) Mol. wt. (kDa) Rg (Å) (± 1) Reference
    Lysozyme (8 mg/mL, 40 mM acetic acid, pH 4.0, 50 mM NaCl) 14.7 13 Voets et al. ( )
    Glucose isomerase (5 mg/mL, 100 mM Tris⋅Cl, pH 8.0, 1 mM MgCl 2) 173 33 Kozak ( )
    Tetraloop‐receptor RNA (PDB ID: 2JYJ) (1 mg/mL, 50 mM Tris⋅Cl, pH 7.0, 150 mM NaCl, 2 mM MgCl 2) 28.2 25 Zuo et al. ( )

Basic Protocol 3: SAXS Data Processing and Analysis

  Materials
  • ATSAS software package (http://www.embl‐hamburg.de/biosaxs/software.html) for the appropriate operating system(s)
  • Sample data files: 2JYJ_APS_SAXS_WAXS_1mgml.dat and 2JYJ_Nanostar_SAXS_1mgml_4h.dat (see Supplemental Files at http://www.currentprotocols.com/protocol/nc0718)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Ali, M., Lipfert, J., Seifert, S., Herschlag, D., and Doniach, S. 2010. The ligand‐free state of the TPP riboswitch: A partially folded RNA structure. J. Mol. Biol. 396:153‐165.
   Baird, N.J. and Ferré‐D'Amaré, A.R. 2010. Idiosyncratically tuned switching behavior of riboswitch aptamer domains revealed by comparative small‐angle X‐ray scattering analysis. RNA 16:598‐609.
   Bernado, P., Mylonas, E., Petoukhov, M.V., Blackledge, M., and Svergun, D.I. 2007. Structural characterization of flexible proteins using small‐angle X‐ray scattering. J. Am. Chem. Soc. 129:5656‐5664.
   Burke, J.E., Sashital, D.G., Zuo, X., Wang, Y.X., and Butcher, S.E. 2012. Structure of the yeast U2/U6 snRNA complex. RNA 18:673‐683.
   Cavaluzzi, M.J. and Borer, P.N. 2004. Revised UV extinction coefficients for nucleoside‐5′‐monophosphates and unpaired DNA and RNA. Nucleic Acids Res. 32:e13.
   Chen, B., Zuo, X., Wang, Y.X., and Dayie, T.K. 2012a. Multiple conformations of SAM‐II riboswitch detected with SAXS and NMR spectroscopy. Nucleic Acids Res. 40:3117‐3130.
   Chen, H., Meisburger, S.P., Pabit, S.A., Sutton, J.L., Webb, W.W., and Pollack, L. 2012b. Ionic strength‐dependent persistence lengths of single‐stranded RNA and DNA. Proc. Natl. Acad. Sci. U.S.A. 109:799‐804.
   Claridge, J.K., Headey, S.J., Chow, J.Y., Schwalbe, M., Edwards, P.J., Jeffries, C.M., Venugopal, H., Trewhella, J., and Pascal, S.M. 2009. A picornaviral loop‐to‐loop replication complex. J. Struct. Biol. 166:251‐262.
   Das, R., Kwok, L.W., Millett, I.S., Bai, Y., Mills, T.T., Jacob, J., Maskel, G.S., Seifert, S., Mochrie, S.G., Thiyagarajan, P., Doniach, S., Pollack, L., and Herschlag, D. 2003. The fastest global events in RNA folding: Electrostatic relaxation and tertiary collapse of the Tetrahymena ribozyme. J. Mol. Biol. 332:311‐319.
   Engelman, D.M., Moore, P.B., and Schoenborn, B.P. 1975. Neutron scattering measurements of separation and shape of proteins in 30S ribosomal subunit of Escherichia coli: S2‐S5, S5‐S8, S3‐S7. Proc. Natl. Acad. Sci. U.S.A. 72:3888‐3892.
   Fang, X., Littrell, K., Yang, X.J., Henderson, S.J., Siefert, S., Thiyagarajan, P., Pan, T., and Sosnick, T.R. 2000. Mg2+‐dependent compaction and folding of yeast tRNAPhe and the catalytic domain of the B. subtilis RNase P RNA determined by small‐angle X‐ray scattering. Biochemistry 39:11107‐11113.
   Ferré D'Amaré, A.R. and Burley, S.K. 1997. Dynamic light‐scattering as a tool for evaluating crystallizability of macromolecules. Meth. Enzymol. 276:157‐166.
   Franke, D. and Svergun, D.I. 2009. DAMMIF, a program for rapid ab‐initio shape determination in small‐angle scattering. J. Appl. Cryst. 42:342‐346.
   Garst, A.D., Heroux, A., Rambo, R.P., and Batey, R.T. 2008. Crystal structure of the lysine riboswitch regulatory mRNA element. J. Biol. Chem. 283:22347‐22351.
   Grishaev, A., Ying, J., Canny, M.D., Pardi, A., and Bax, A. 2008. Solution structure of tRNAVal from refinement of homology model against residual dipolar coupling and SAXS data. J. Biomol. NMR 42:99‐109.
   Hammond, J.A., Rambo, R.P., Filbin, M.E., and Kieft, J.S. 2009. Comparison and functional implications of the 3D architectures of viral tRNA‐like structures. RNA 15:294‐307.
   Harmanci, A.O., Sharma, G., and Mathews, D.H. 2011. TurboFold: Iterative probabilistic estimation of secondary structures for multiple RNA sequences. BMC Bioinformatics 12:108.
   Huang, T.C., Toraya, H., Blanton, T.N., and Wu, Y. 1993. X‐ray‐powder diffraction analysis of silver behenate, a possible low‐angle diffraction standard. J. Appl. Crystallogr. 26:180‐184.
   Jachimska, B., Wasilewska, M., and Adamczyk, Z. 2008. Characterization of globular protein solutions by dynamic light scattering, electrophoretic mobility, and viscosity measurements. Langmuir 24:6866‐6872.
   Jonikas, M.A., Radmer, R.J., Laederach, A., Das, R., Pearlman, S., Herschlag, D., and Altman, R.B. 2009. Coarse‐grained modeling of large RNA molecules with knowledge‐based potentials and structural filters. RNA 15:189‐199.
   Juan, V. and Wilson, C. 1999. RNA secondary structure prediction based on free energy and phylogenetic analysis. J. Mol. Biol. 289:935‐947.
   Kazantsev, A.V., Rambo, R.P., Karimpour, S., Santalucia, J. Jr., Tainer, J.A., and Pace, N.R. 2011. Solution structure of RNase P RNA. RNA 17:1159‐1171.
   Konarev, P.V., Volkov, V.V., Sokolova, A.V., Koch, M.H.J., and Svergun, D.I. 2003. PRIMUS: A Windows PC‐based system for small‐angle scattering data analysis. J. Appl. Crystallogr. 36:1277‐1282.
   Kozak, M. 2005. Direct comparison of the crystal and solution structure of glucose/xylose isomerase from Streptomyces rubigenosus. Protein Pept. Lett. 12:547‐550.
   Kozin, M.B. and Svergun, D.I. 2001. Automated matching of high‐ and low‐resolution structural models. J. Appl. Crystallogr. 34:33‐41.
   Lake, J.A. 1967. Yeast transfer RNA: A small‐angle X‐ray study. Science 156:1371‐1373.
   Lescoute, A. and Westhof, E. 2006. Topology of three‐way junctions in folded RNAs. RNA 12:83‐93.
   Lipfert, J., Das, R., Chu, V.B., Kudaravalli, M., Boyd, N., Herschlag, D., and Doniach, S. 2007. Structural transitions and thermodynamics of a glycine‐dependent riboswitch from Vibrio cholerae. J. Mol. Biol. 365:1393‐1406.
   Lipfert, J., Ouellet, J., Norman, D.G., Doniach, S., and Lilley, D.M. 2008. The complete VS ribozyme in solution studied by small‐angle X‐ray scattering. Structure 16:1357‐1367.
   Lipfert, J., Sim, A.Y., Herschlag, D., and Doniach, S. 2010. Dissecting electrostatic screening, specific ion binding, and ligand binding in an energetic model for glycine riboswitch folding. RNA 16:708‐719.
   Low, J.T. and Weeks, K.M. 2010. SHAPE‐directed RNA secondary structure prediction. Methods 52:150‐158.
   Mathews, D.H., Disney, M.D., Childs, J.L., Schroeder, S.J., Zuker, M., and Turner, D.H. 2004. Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc. Natl. Acad. Sci. U.S.A. 101:7287‐7292.
   Milligan, J.F. and Uhlenbeck, O.C. 1989. Synthesis of small RNAs using T7 RNA polymerase. Meth. Enzymol. 180:51‐62.
   Miyazaki, Y., Irobalieva, R.N., Tolbert, B.S., Smalls‐Mantey, A., Iyalla, K., Loeliger, K., D'Souza, V., Khant, H., Schmid, M.F., Garcia, E.L., Telesnitsky, A., Chiu, W., and Summers, M.F. 2010. Structure of a conserved retroviral RNA packaging element by NMR spectroscopy and cryo‐electron tomography. J. Mol. Biol. 404:751‐772.
   Nelissen, F.H., Leunissen, E.H., van de Laar, L., Tessari, M., Heus, H.A., and Wijmenga, S.S. 2012. Fast production of homogeneous recombinant RNA‐Towards large‐scale production of RNA. Nucleic Acids Res. 40:e102
   Ninio, J., Luzzati, V., and Yaniv, M. 1972. Comparative small‐angle X‐ray scattering studies on unacylated, acylated and cross‐linked Escherichia coli transfer RNA I Val. J. Mol. Biol. 71:217‐229.
   Nollmann, M., Stark, W.M., and Byron, O. 2004. Low‐resolution reconstruction of a synthetic DNA holiday junction. Biophys. J. 86:3060‐3069.
   Parisien, M. and Major, F. 2008. The MC‐Fold and MC‐Sym pipeline infers RNA structure from sequence data. Nature 452:51‐55.
   Pelikan, M., Hura, G.L., and Hammel, M. 2009. Structure and flexibility within proteins as identified through small angle X‐ray scattering. Gen. Physiol. Biophys. 28:174‐189.
   Pereira, M.J., Behera, V., and Walter, N.G. 2010. Nondenaturing purification of co‐transcriptionally folded RNA avoids common folding heterogeneity. PLoS One 5:e12953.
   Pollack, L. 2011. Time resolved SAXS and RNA folding. Biopolymers 95:543‐549.
   Pollack, L. and Doniach, S. 2009. Time‐resolved X‐ray scattering and RNA folding. Meth. Enzymol. 469:253‐268.
   Price, S.R., Ito, N., Oubridge, C., Avis, J.M., and Nagai, K. 1995. Crystallization of RNA‐protein complexes. I. Methods for the large‐scale preparation of RNA suitable for crystallographic studies. J. Mol. Biol. 249:398‐408.
   Putnam, C.D., Hammel, M., Hura, G.L., and Tainer, J.A. 2007. X‐ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q. Rev. Biophys. 40:191‐285.
   Rambo, R.P. and Tainer, J.A. 2010. Improving small‐angle X‐ray scattering data for structural analyses of the RNA world. RNA 16:638‐646.
   Roh, J.H., Guo, L., Kilburn, J.D., Briber, R.M., Irving, T., and Woodson, S.A. 2010. Multistage collapse of a bacterial ribozyme observed by time‐resolved small‐angle X‐ray scattering. J. Am. Chem. Soc. 132:10148‐10154.
   Russell, R., Millett, I.S., Doniach, S., and Herschlag, D. 2000. Small angle X‐ray scattering reveals a compact intermediate in RNA folding. Nat. Struct. Biol. 7:367‐370.
   Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   Schneidman‐Duhovny, D., Hammel, M., and Sali, A. 2010. FoXS: A Web server for rapid computation and fitting of SAXS profiles. Nucleic Acids Res. 38:W540‐W544.
   Scott, L.G. and Hennig, M. 2008. RNA structure determination by NMR. Meth. Mol. Biol. 452:29‐61.
   Shpungin, I.L., Perevozchikov, V.A., Serdiuk, I.N., and Zaccai, G. 1980. Isolation and physical study of the 13S fragment of 16S RNA and its complex with ribosomal protein S4. Mol. Biol. 14:939‐950.
   Soliman, M., Jungnickel, B.J., and Meister, E. 1998. Stable desmearing of slit‐collimated SAXS patterns by adequate numerical conditioning. Acta Crystallogr. A 54:675‐681.
   Stark, H. and Luhrmann, R. 2006. Cryo‐electron microscopy of spliceosomal components. Annu. Rev. Biophys. Biomol. Struct. 35:435‐457.
   Suhre, K. and Sanejouand, Y.H. 2004. ElNemo: A normal mode Web server for protein movement analysis and the generation of templates for molecular replacement. Nucleic Acids Res. 32:W610‐W614.
   Svergun, D.I. 1992. Determination of the regularization parameter in indirect‐transform methods using perceptual criteria. J. Appl. Cryst. 25:495‐503.
   Svergun, D.I., Burkhardt, N., Pedersen, J.S., Koch, M.H., Volkov, V.V., Kozin, M.B., Meerwink, W., Stuhrmann, H.B., Diedrich, G., and Nierhaus, K.H. 1997. Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation. II. A model of the ribosome and its RNA at 3.5 nm resolution. J. Mol. Biol. 271:602‐618.
   Voets, I.K., Cruz, W.A., Moitzi, C., Lindner, P., Areas, E.P., and Schurtenberger, P. 2010. DMSO‐induced denaturation of hen egg white lysozyme. J. Phycs. Chem. B 114:11875‐11883.
   Walker, S.C., Avis, J.M., and Conn, G.L. 2003. General plasmids for producing RNA in vitro transcripts with homogeneous ends. Nucleic Acids Res. 31:e82.
   Wang, Y.X., Zuo, X., Wang, J., Yu, P., and Butcher, S.E. 2010. Rapid global structure determination of large RNA and RNA complexes using NMR and small‐angle X‐ray scattering. Methods 52:180‐191.
   Yang, S., Parisien, M., Major, F., and Roux, B. 2010. RNA structure determination using SAXS data. J. Phys. Chem. B 114:10039‐10048.
   Zhang, F., Ilavsky, J., Long, G.G., Quintana, J.P.G., Allen, A.J., and Jemian, P.R. 2010. Glassy carbon as an absolute intensity calibration standard for small‐angle scattering. Metall. Mater. Trans. A 41A:1151‐1158.
   Zuker, M. 2003. MFold Web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31:3406‐3415.
   Zuo, X., Wang, J., Foster, T.R., Schwieters, C.D., Tiede, D.M., Butcher, S.E., and Wang, Y.X. 2008. Global molecular structure and interfaces: Refining an RNA:RNA complex structure using solution X‐ray scattering data. J. Am. Chem. Soc. 130:3292‐3293.
   Zuo, X., Wang, J., Yu, P., Eyler, D., Xu, H., Starich, M.R., Tiede, D.M., Simon, A.E., Kasprzak, W., Schwieters, C.D., Shapiro, B.A., and Wang, Y.X. 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. U.S.A. 107:1385‐1390.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Supplementary Material

Sample data files for the tetraloop-receptor RNA complex: nc0718.zip