Skip to main content
SpringerLink
Log in
Book cover

RNA Spectroscopy pp 165–188Cite as

Small-Angle Neutron Scattering of RNA–Protein Complexes

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2113))

Abstract

Small-angle neutron scattering (SANS) provides structural information on biomacromolecules and their complexes in dilute solutions at the nanometer length scale. The overall dimensions, shapes, and interactions can be probed and compared to information obtained by complementary structural biology techniques such as crystallography, NMR, and EM. SANS, in combination with solvent H2O/D2O exchange and/or deuteration, is particularly well suited to probe the internal structure of RNA–protein (RNP) complexes since neutrons are more sensitive than X-rays to the difference in scattering length densities of proteins and RNA, with respect to an aqueous solvent. In this book chapter we provide a practical guide on how to carry out SANS experiments on RNP complexes, as well as possibilities of data analysis and interpretation.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Jacrot B, Chauvin C, Witz J (1977) Comparative neutron small-angle scattering study of small spherical RNA viruses. Nature 266:417–421

    CAS  PubMed  Google Scholar 

  2. Giegé R, Jacrot B, Moras D, Thierry J-C, Zaccai G (1977) A neutron investigation of yeast valyl-tRNA synthetase interaction with tRNAs. Nucleic Acids Res 7(7):2421–2427

    Google Scholar 

  3. Dessen P, Blanquet S, Zaccai G, Jacrot B (1978) Antico-operative binding of initiator transfer RNAMet to methionyl-transfer RNA synthetase from Escherichia coli: neutron scattering studies. J Mol Biol 126:293–313

    CAS  PubMed  Google Scholar 

  4. Hoppe W, May R, Stoeckel P, Lorenz S, Erdmann VA, Wittmann HG, Crespi HL, Katz JJ, Ibel K (1976) Neutron scattering measurements with the label triangulation method on the 50S subunit of E. coli ribosomes. In: Schoenborn BP (ed) Neutron scattering for the analysis of biological structures, Brookhaven, 1975. Brookhaven symposia in biology. Brookhaven National Laboratory, Upton, pp IV38–IV48

    Google Scholar 

  5. Stuhrmann HB, Haas J, Ibel K, Wolf B, Koch MH, Parfait R, Crichton RR (1976) New low resolution model for 50S subunit of Escherichia coli ribosomes. Proc Natl Acad Sci U S A 73(7):2379–2383

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Engelman DM, Moore BP, Schoenborn BP (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(10):3888–3892

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Engelman DM, Moore PB (1972) A new method for the determination of biological quaternary structure by neutron scattering. Proc Natl Acad Sci USA 69:1997–1999

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Hoppe W (1972) A new X-ray method for the determination of the quaternary structure of protein complexes. Isr J Chem 10:321–333

    CAS  Google Scholar 

  9. Stuhrmann HB, Kirste RG (1965) Elimination der intrapartikulaeren Untergrundstreuung bei der Roentgenkleinwinkelstreuung an kompakten Teilen (Proteinen). Z Phys Chem 46(3–4):247–250

    CAS  Google Scholar 

  10. Pardon JF, Worcester DL, Wooley JC, Tatchell K, Van Holde KE, Richards BM (1975) Low-angle neutron scattering from chromatin subunit particles. Nucleic Acids Res 2(11):2163–2176

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Baldwin JP, Boseley PG, Bradbury EM, Ibel K (1975) The subunit structure of the eukaryotic chromosome. Nature 253:245–249

    CAS  PubMed  Google Scholar 

  12. Bram S, Butler-Browne G, Baudy P, Ibel K (1975) Quaternary structure of chromatin. Proc Natl Acad Sci U S A 72(3):1043–1045

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Jacrot B (1976) The study of biological structures by neurtron scattering from solution. Rep Prog Phys 39(10):911–953

    CAS  Google Scholar 

  14. Zaccai G, Jacrot B (1983) Small angle neutron scattering. Annu Rev Biophys Biomol Struct 12:139–157

    CAS  Google Scholar 

  15. Glatter O, Kratky O (1982) Small angle X-ray scattering. Academic Press, London

    Google Scholar 

  16. Kratky O (1972) Recent advances and applications of diffuse X-ray small-angle scattering on biopolymers in dilute solutions. Q Rev Biophys 5(4):481–537

    CAS  PubMed  Google Scholar 

  17. Svergun DI, Barberato C, Koch MHJ (1995) CRYSOL—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Crystallogr 28:768–773

    CAS  Google Scholar 

  18. Svergun DI, Richard S, Koch MHJ, Sayers Z, Kuprin S, Zaccai G (1998) Protein hydration in solution: experimental observation by X-ray and neutron scattering. Proc Natl Acad Sci USA 95(5):2267–2272

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Svergun DI, Nierhaus KH (2000) A map of protein-rRNA distribution in the 70S Escherichia coli ribosome. J Biol Chem 275(19):14432–14439

    CAS  PubMed  Google Scholar 

  20. Svergun DI (1999) Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys J 76(6):2879–2886

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Mertens HDT, Svergun DI (2017) Combining NMR and small angle X-ray scattering for the study of biomolecular structure and dynamics. Arch Biochem Biophys 628:33–41. https://doi.org/10.1016/j.abb.2017.05.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hennig J, Sattler M (2014) The dynamic duo: combining NMR and small angle scattering in structural biology. Protein Sci 23:669. https://doi.org/10.1002/pro.2467

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Carlomagno T (2014) Present and future of NMR for RNA-protein complexes: a perspective of integrated structural biology. J Magn Reson 241:126–136

    CAS  PubMed  Google Scholar 

  24. Madl T, Gabel F, Sattler M (2011) NMR and small-angle scattering-based structural analysis of protein complexes in solution. J Struct Biol 173(3):472–482. https://doi.org/10.1016/j.jsb.2010.11.004

    Article  CAS  PubMed  Google Scholar 

  25. Svergun DI, Koch MHJ, Timmins PA, May RP (2013) Small angle X-ray and neutron scattering from solutions of biological macromolecules. IUCr monographs on crystallography, vol 19. Oxford University Press, Oxford

    Google Scholar 

  26. Gutberlet T, Rücker U, Brückel T (2017) Towards compact accelerator driven neutronsources for Europe. Neutron News 28(3):20–25. https://doi.org/10.1080/10448632.2017.1342486

    Article  Google Scholar 

  27. Rauch H, Waschkowski W (2001) Neutron scattering lengths. In: Dianoux A-J, Lander G (eds) Neutron data booklet. Institut Laue-Langevin, Grenoble

    Google Scholar 

  28. Jacrot B, Zaccai G (1981) Determination of molecular weight by neutron scattering. Biopolymers 20:2413–2426

    CAS  Google Scholar 

  29. Jeffries CM, Graewert MA, Blanchet CE, Langley DB, Whitten AE, Svergun DI (2016) Preparing monodisperse macromolecular samples for successful biological small-angle X-ray and neutron-scattering experiments. Nat Protoc 11(11):2122–2153. https://doi.org/10.1038/nprot.2016.113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Jacques DA, Trewhella J (2010) Small-angle scattering for structural biology—expanding the frontier while avoiding the pitfalls. Protein Sci 19:642–657

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Putnam CD, Hammel M, Hura GL, Tainer JA (2007) X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q Rev Biophys 40(3):191–285

    CAS  PubMed  Google Scholar 

  32. Zaccai NR, Serdyuk IN, Zaccai J (2017) Methods in molecular biophysics: structure, dynamics, function for biology and medicine, 2nd edn. Cambridge University Press, Cambridge. https://doi.org/10.1017/9781107297227

    Book  Google Scholar 

  33. Philo JS (2006) Is any measurement method optimal for all aggregate sizes and types? AAPS J 8(3):E564–E571. https://doi.org/10.1208/aapsj080365

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Porterfield JZ, Zlotnick A (2010) A simple and general method for determining the protein and nucleic acid content of viruses by UV absorbance. Virology 407(2):281–288. https://doi.org/10.1016/j.virol.2010.08.015

    Article  CAS  PubMed  Google Scholar 

  35. Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 15(21):8783–8798

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Haertlein M, Moulin M, Devos JM, Laux V, Dunne O, Trevor Forsyth V (2016) Biomolecular deuteration for neutron structural biology and dynamics. In: Kelman Z (ed) Methods in enzymology, vol 566. Academic Press, pp 113–157. https://doi.org/10.1016/bs.mie.2015.11.001

    Google Scholar 

  37. Hattori A, Crespi HL, Katz JJ (1965) Effect of side-chain deuteration on protein stability. Biochemistry 4(7):1213–1225

    CAS  PubMed  Google Scholar 

  38. Makhatadze GI, Clore GM, Gronenborn AM (1995) Solvent isotope effect and protein stability. Nat Struct Biol 2(10):852–855

    CAS  PubMed  Google Scholar 

  39. Rio DC (2012) Filter-binding assay for analysis of RNA–protein interactions. Cold Spring Harb Protoc 2012(10):pdb.prot071449

    PubMed  Google Scholar 

  40. Ryder SP, Recht MI, Williamson JR (2008) Quantitative analysis of protein-RNA interactions by gel mobility shift. Methods Mol Biol (Clifton, NJ) 488:99–115. https://doi.org/10.1007/978-1-60327-475-3_7

    Article  CAS  Google Scholar 

  41. Recht MI, Ryder SP, Williamson JR (2008) Monitoring assembly of ribonucleoprotein complexes by isothermal titration calorimetry. Methods Mol Biol (Clifton, NJ) 488:117–127. https://doi.org/10.1007/978-1-60327-475-3_8

    Article  CAS  Google Scholar 

  42. Lapinaite A, Simon B, Skjaerven L, Rakwalska-Bange M, Gabel F, Carlomagno T (2013) The structure of the box C/D enzyme reveals regulation of RNA methylation. Nature 502(7472):519–523. https://doi.org/10.1038/nature12581. http://www.nature.com/nature/journal/v502/n7472/abs/nature12581.html#supplementary-information

    Article  CAS  PubMed  Google Scholar 

  43. Feigin LA, Svergun DI (1987) Structure analysis by small-angle X-ray and neutron scattering. Plenum Press, New York

    Google Scholar 

  44. Guinier A, Fournet G (1955) Small angle scattering of X-rays. Wiley, New York

    Google Scholar 

  45. Voss NR, Gerstein M (2005) Calculation of standard atomic volumes for RNA and comparison with proteins: RNA is packed more tightly. J Mol Biol 346:477–492

    CAS  PubMed  Google Scholar 

  46. Sarachan KL, Curtis JE, Krueger S (2014) Small-angle scattering contrast calculator for protein and nucleic acid complexes in solution. J Appl Crystallogr 46:1889–1893

    Google Scholar 

  47. Whitten AE, Cai S, Trewhella J (2008) MULCh: modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies. J Appl Crystallog 41(1):222–226. https://doi.org/10.1107/S0021889807055136

    Article  CAS  Google Scholar 

  48. Hennig J, Militti C, Popowicz GM, Wang I, Sonntag M, Geerlof A, Gabel F, Gebauer F, Sattler M (2014) Structural basis for the assembly of the Sxl-Unr translation regulatory complex. Nature 515(7526):287–290. https://doi.org/10.1038/nature13693

    Article  CAS  PubMed  Google Scholar 

  49. Mahieu E, Gabel F (2018) Biological small-angle neutron scattering: recent results and development. Acta Crystallogr Sect D 74(8):715–726. https://doi.org/10.1107/S2059798318005016

    Article  CAS  Google Scholar 

  50. Konarev PV, Volkov VV, Sokolova AV, Koch MHJ, Svergun DI (2003) PRIMUS: a Windows PC-based system for small-angle scattering data analysi. J Appl Crystallogr 36:1277–1282

    CAS  Google Scholar 

  51. Gabel F, Bicout D, Lehnert U, Tehei M, Weik M, Zaccai G (2002) Protein dynamics studied by neutron scattering. Q Rev Biophys 35(4):327–367

    CAS  PubMed  Google Scholar 

  52. Guinier A (1939) La diffraction des Rayons X aux Tres Faibles Angles: Applications a l'Etude des Phenomenes Ultra-microscopiques. Annal Phys (Paris) 12:161–236

    CAS  Google Scholar 

  53. Kim Henry S, Martel A, Girard E, Moulin M, Härtlein M, Madern D, Blackledge M, Franzetti B, Gabel F (2016) SAXS/SANS on supercharged proteins reveals residue-specific modifications of the hydration shell. Biophys J 110(10):2185–2194. https://doi.org/10.1016/j.bpj.2016.04.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Kim HS, Gabel F (2015) Uniqueness of quasi-atomic bio-macromolecularmodels derived from small angle scattering data. Acta Crystallogr D Biol Crystallogr 71(Pt 1):57–66

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Jacrot B (1976) The study of biological structures by neutron scattering from solution. Rep Prog Phys 39(10):911–953

    CAS  Google Scholar 

  56. Grotwinkel JT, Wild K, Segnitz B, Sinning I (2014) SRP RNA remodeling by SRP68 explains its role in protein translocation. Science 344(6179):101. https://doi.org/10.1126/science.1249094

    Article  CAS  PubMed  Google Scholar 

  57. Wang Y-X, Zuo X, Wang J, Yu P, Butcher SE (2010) Rapid global structure determination of large RNA and RNA complexes using NMR and small-angle X-ray scattering. Methods (San Diego, CA) 52(2):180–191. https://doi.org/10.1016/j.ymeth.2010.06.009

    Article  CAS  Google Scholar 

  58. Falb M, Amata I, Gabel F, Simon B, Carlomagno T (2010) Structure of the K-turn U4 RNA: a combined NMR and SANS study. Nucleic Acids Res 38(18):6274–6285

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Grishaev A, Wu J, Trewhella J, Bax A (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

    CAS  PubMed  Google Scholar 

  60. Trabuco LG, Villa E, Mitra K, Frank J, Schulten K (2008) Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure (London, England : 1993) 16(5):673–683. https://doi.org/10.1016/j.str.2008.03.005

    Article  CAS  Google Scholar 

  61. Karaca E, Rodrigues JPGLM, Graziadei A, Bonvin AMJJ, Carlomagno T (2017) M3: an integrative framework for structure determination of molecular machines. Nat Methods 14:897. https://doi.org/10.1038/nmeth.4392. https://www.nature.com/articles/nmeth.4392#supplementary-information

    Article  CAS  PubMed  Google Scholar 

  62. Sonntag M, Jagtap PKA, Simon B, Appavou M-S, Geerlof A, Stehle R, Gabel F, Hennig J, Sattler M (2017) Segmental, domain-selective perdeuteration and small-angle neutron scattering for structural analysis of multi-domain proteins. Angew Chem Int Ed 56(32):9322–9325. https://doi.org/10.1002/anie.201702904

    Article  CAS  Google Scholar 

  63. Michel E, Allain FHT (2015) Selective amino acid segmental labeling of multi-domain proteins. In: Kelman Z (ed) Methods in enzymology, vol 565. Academic Press, New York, pp 389–422. https://doi.org/10.1016/bs.mie.2015.05.028

    Chapter  Google Scholar 

  64. Freiburger L, Sonntag M, Hennig J, Li J, Zou P, Sattler M (2015) Efficient segmental isotope labeling of multi-domain proteins using Sortase a. J Biomol NMR 63(1):1–8. https://doi.org/10.1007/s10858-015-9981-0

    Article  CAS  PubMed  Google Scholar 

  65. Michel E, Skrisovska L, Wüthrich K, Allain FH (2013) Amino acid-selective segmental isotope labeling of multidomain proteins for structural biology. Chembiochem 14(4):457–466

    CAS  PubMed  Google Scholar 

  66. Duss O, Lukavsky PJ, Allain FH (2012) Isotope labeling and segmental labeling of larger RNAs for NMR structural studies. Adv Exp Med Biol 992:121–144

    CAS  PubMed  Google Scholar 

  67. Nelissen FHT, van Gammeren AJ, Tessari M, Girard FC, Heus HA, Wijmenga SS (2008) Multiple segmental and selective isotope labeling of large RNA for NMR structural studies. Nucleic Acids Res 36(14):e89–e89. https://doi.org/10.1093/nar/gkn397

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Tzakos AG, Easton LE, Lukavsky PJ (2007) Preparation of large RNA oligonucleotides with complementary isotope-labeled segments for NMR structural studies. Nat Protoc 2:2139. https://doi.org/10.1038/nprot.2007.306

    Article  CAS  PubMed  Google Scholar 

  69. Nemethy G, Scheraga HA (1962) Structure of water and hyrdophobic bonding in proteins. I. A model for the thermodynamic properties in liquid water. J Chem Phys 36(12):3382–3417

    CAS  Google Scholar 

Download references

Acknowledgments

FG acknowledges funding from the French Agence Nationale de la Recherche (grant ANR-15-CE11-0026-01 “PROTstretch”). TC acknowledges funding from the DFG (grants CA294/3-2 and CA294/11-1).

Author information

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

    Audrone Lapinaite

  2. Centre for Biomolecular Drug Research, Leibniz University Hannover, Hannover, Germany

    Teresa Carlomagno

  3. Helmholtz Centre for Infection Research, Group of Structural Chemistry, Braunschweig, Germany

    Teresa Carlomagno

  4. Univ. Grenoble Alpes, CEA, CNRS, IBS, Grenoble, France

    Frank Gabel

Authors
  1. Audrone Lapinaite
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Teresa Carlomagno
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Frank Gabel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Frank Gabel .

Editor information

Editors and Affiliations

  1. Université de Paris, Paris, France, Laboratoire Léon Brillouin LLB, CEA, CNRS UMR12, Université Paris Saclay, Gif-sur-Yvette, France

    Véronique Arluison

  2. Synchrotron SOLEIL L’Orme des Merisiers Saint Aubin, Gif-sur-Yvette, France

    Frank Wien

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Lapinaite, A., Carlomagno, T., Gabel, F. (2020). Small-Angle Neutron Scattering of RNA–Protein Complexes. In: Arluison, V., Wien, F. (eds) RNA Spectroscopy. Methods in Molecular Biology, vol 2113. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0278-2_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0278-2_13

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0277-5

  • Online ISBN: 978-1-0716-0278-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation