Soaking Hexammine Cations into RNA Crystals to Obtain Derivatives for Phasing Diffraction Data

  • Robert T. BateyEmail author
  • Jeffrey S. KieftEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1320)


Solving a novel RNA structure by x-ray crystallography requires a means to obtain initial phase estimates. This is a challenge because many of the tools available for solving protein structures are not available for RNA. We have developed a reliable means to use hexammine cations to address this challenge. The process involves engineering the RNA to introduce a reliable hexammine binding site into the structure, then soaking crystals of these RNAs with an iridium (III) or cobalt (III) compound in a “directed soaking” strategy. Diffraction data obtained from these crystals then can be used in SAD or MAD phasing. In many cases, suitable derivatives can be obtained by soaking the hexammine into RNA crystals that have not been engineered. Considerations for using this method and example protocols are presented.

Key words

RNA Hexammine Structure solution Heavy atoms Iridium 



The authors thank current and former members of our labs for thoughtful discussions and technical assistance and David Costantino for critical reading of this manuscript. R.T.B. is supported by NIH grants GM073850 and GM083953. J.S.K. is supported by NIH grants GM097333 and GM081346. J.S.K. is an Early Career Scientist of the Howard Hughes Medical Institute.


  1. 1.
    McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    McCoy AJ (2007) Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr D Biol Crystallogr 63:32–41PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Doublie S (1997) Preparation of selenomethionyl proteins for phase determination. Methods Enzymol 276:523–530PubMedCrossRefGoogle Scholar
  4. 4.
    Hendrickson WA, Horton JR, LeMaster DM (1990) Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. EMBO J 9:1665–1672PubMedCentralPubMedGoogle Scholar
  5. 5.
    Robertson MP, Scott WG (2007) The structural basis of ribozyme-catalyzed RNA assembly. Science 315:1549–1553PubMedCrossRefGoogle Scholar
  6. 6.
    Robertson MP, Scott WG (2008) A general method for phasing novel complex RNA crystal structures without heavy-atom derivatives. Acta Crystallogr D Biol Crystallogr 64:738–744PubMedCentralCrossRefGoogle Scholar
  7. 7.
    Robertson MP, Chi YI, Scott WG (2010) Solving novel RNA structures using only secondary structural fragments. Methods 52:168–172PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Brandt G, Carrasco N, Huang Z (2006) Efficient substrate cleavage catalyzed by hammerhead ribozymes derivatized with selenium for X-ray crystallography. Biochemistry 45:8972–8977PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Golden BL (2000) Heavy atom derivatives of RNA. Methods Enzymol 317:124–132PubMedCrossRefGoogle Scholar
  10. 10.
    Jiang J, Sheng J, Carrasco N, Huang Z (2007) Selenium derivatization of nucleic acids for crystallography. Nucleic Acids Res 35:477–485PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Carrasco N, Buzin Y, Tyson E, Halpert E, Huang Z (2004) Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion. Nucleic Acids Res 32:1638–1646PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Hobartner C, Micura R (2004) Chemical synthesis of selenium-modified oligoribonucleotides and their enzymatic ligation leading to an U6 SnRNA stem-loop segment. J Am Chem Soc 126:1141–1149PubMedCrossRefGoogle Scholar
  13. 13.
    Hobartner C, Rieder R, Kreutz C, Puffer B, Lang K, Polonskaia A, Serganov A, Micura R (2005) Syntheses of RNAs with up to 100 nucleotides containing site-specific 2'-methylseleno labels for use in X-ray crystallography. J Am Chem Soc 127:12035–12045PubMedCrossRefGoogle Scholar
  14. 14.
    Salon J, Sheng J, Jiang J, Chen G, Caton-Williams J, Huang Z (2007) Oxygen replacement with selenium at the thymidine 4-position for the Se base pairing and crystal structure studies. J Am Chem Soc 129:4862–4863PubMedCrossRefGoogle Scholar
  15. 15.
    Sheng J, Jiang J, Salon J, Huang Z (2007) Synthesis of a 2'-Se-thymidine phosphoramidite and its incorporation into oligonucleotides for crystal structure study. Org Lett 9:749–752PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Baugh C, Grate D, Wilson C (2000) 2.8 A crystal structure of the malachite green aptamer. J Mol Biol 301:117–128PubMedCrossRefGoogle Scholar
  17. 17.
    Kieft JS, Zhou K, Grech A, Jubin R, Doudna JA (2002) Crystal structure of an RNA tertiary domain essential to HCV IRES-mediated translation initiation. Nat Struct Biol 9:370–374PubMedGoogle Scholar
  18. 18.
    Martick M, Scott WG (2006) Tertiary contacts distant from the active site prime a ribozyme for catalysis. Cell 126:309–320PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Golden BL, Gooding AR, Podell ER, Cech TR (1996) X-ray crystallography of large RNAs: heavy-atom derivatives by RNA engineering. RNA 2:1295–1305PubMedCentralPubMedGoogle Scholar
  20. 20.
    Wedekind JE, McKay DB (2000) Purification, crystallization, and X-ray diffraction analysis of small ribozymes. Methods Enzymol 317:149–168PubMedCrossRefGoogle Scholar
  21. 21.
    Keel AY, Rambo RP, Batey RT, Kieft JS (2007) A general strategy to solve the phase problem in RNA crystallography. Structure 15:761–772PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Masquida B, Westhof E (2000) On the wobble GoU and related pairs. RNA 6:9–15PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Varani G, McClain WH (2000) The G x U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems. EMBO Rep 1:18–23PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Kieft JS (1997). Structure and thermodynamics of a metal ion binding site in the RNA major groove: cobalt (III) hexammine as a probe. [Thesis]. Type, University of California, Berkeley, USAGoogle Scholar
  25. 25.
    Cate JH, Doudna JA (1996) Metal-binding sites in the major groove of a large ribozyme domain. Structure 4:1221–1229PubMedCrossRefGoogle Scholar
  26. 26.
    Colmenarejo G, Tinoco I Jr (1999) Structure and thermodynamics of metal binding in the P5 helix of a group I intron ribozyme. J Mol Biol 290:119–135PubMedCrossRefGoogle Scholar
  27. 27.
    Montange RK, Batey RT (2006) Structure of the S-adenosylmethionine riboswitch regulatory mRNA element. Nature 441:1172–1175PubMedCrossRefGoogle Scholar
  28. 28.
    Stefan LR, Zhang R, Levitan AG, Hendrix DK, Brenner SE, Holbrook SR (2006) MeRNA: a database of metal ion binding sites in RNA structures. Nucleic Acids Res 34:D131–D134PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Batey RT, Gilbert SD, Montange RK (2004) Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine. Nature 432:411–415PubMedCrossRefGoogle Scholar
  30. 30.
    Cate JH, Gooding AR, Podell E, Zhou K, Golden BL, Kundrot CE, Chech TR, Doudna JA (1996) Crystal structure of a group I ribozyme domain: principles of RNA packing. Science 273:1678–1685PubMedCrossRefGoogle Scholar
  31. 31.
    Cochrane JC, Lipchock SV, Strobel SA (2007) Structural investigation of the GlmS ribozyme bound to its catalytic cofactor. Chem Biol 14:97–105PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Kazantsev AV, Krivenko AA, Harrington DJ, Holbrook SR, Adams PD, Pace NR (2005) Crystal structure of a bacterial ribonuclease P RNA. Proc Natl Acad Sci U S A 102:13392–13397PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Toor N, Keating KS, Taylor SD, Pyle AM (2008) Crystal structure of a self-spliced group II intron. Science 320:77–82PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Pfingsten JS, Costantino DA, Kieft JS (2006) Structural basis for ribosome recruitment and manipulation by a viral IRES RNA. Science 314:1450–1454PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Costantino DA, Pfingsten JS, Rambo RP, Kieft JS (2008) tRNA-mRNA mimicry drives translation initiation from a viral IRES. Nat Struct Mol Biol 15:57–64PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Peselis A, Serganov A (2012) Structural insights into ligand binding and gene expression control by an adenosylcobalamin riboswitch. Nat Struct Mol Biol 19:1182–1184PubMedCrossRefGoogle Scholar
  37. 37.
    Smith KD, Lipchock SV, Ames TD, Wang J, Breaker RR, Strobel SA (2009) Structural basis of ligand binding by a c-di-GMP riboswitch. Nat Struct Mol Biol 16:1218–1223PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Garst AD, Heroux A, Rambo RP, Batey RT (2008) Crystal structure of the lysine riboswitch regulatory mRNA element. J Biol Chem 283:22347–22351PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Huang L, Serganov A, Patel DJ (2010) Structural insights into ligand recognition by a sensing domain of the cooperative glycine riboswitch. Mol Cell 40:774–786PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Ban N, Nissen P, Hansen J, Moore PB, Steitz TA (2000) The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 289:905–920PubMedCrossRefGoogle Scholar
  41. 41.
    Cate JH, Yusupov MM, Yusupova GZ, Earnest TN, Noller HF (1999) X-ray crystal structures of 70S ribosome functional complexes. Science 285:2095–2104PubMedCrossRefGoogle Scholar
  42. 42.
    Clemons WM Jr, May JL, Wimberly BT, McCutcheon JP, Capel MS, Ramakrishnan V (1999) Structure of a bacterial 30S ribosomal subunit at 5.5 A resolution. Nature 400:833–840PubMedCrossRefGoogle Scholar
  43. 43.
    Gilbert SD, Rambo RP, Van Tyne D, Batey RT (2008) Structure of the SAM-II riboswitch bound to S-adenosylmethionine. Nat Struct Mol Biol 15:177–182PubMedCrossRefGoogle Scholar
  44. 44.
    Edwards AL, Reyes FE, Heroux A, Batey RT (2010) Structural basis for recognition of S-adenosylhomocysteine by riboswitches. RNA 16:2144–2155PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Ren A, Rajashankar KR, Patel DJ (2012) Fluoride ion encapsulation by Mg2+ ions and phosphates in a fluoride riboswitch. Nature 486:85–89PubMedCentralPubMedGoogle Scholar
  46. 46.
    Serganov A, Huang L, Patel DJ (2009) Coenzyme recognition and gene regulation by a flavin mononucleotide riboswitch. Nature 458:233–237PubMedCentralPubMedCrossRefGoogle Scholar
  47. 47.
    Johnson JE Jr, Reyes FE, Polaski JT, Batey RT (2012) B12 cofactors directly stabilize an mRNA regulatory switch. Nature 492:133–137PubMedCentralPubMedCrossRefGoogle Scholar
  48. 48.
    Kieft JS, Costantino DA, Filbin ME, Hammond J, Pfingsten JS (2007) Structural methods for studying IRES function. Methods Enzymol 430:333–371PubMedCrossRefGoogle Scholar
  49. 49.
    Edwards AL, Garst AD, Batey RT (2009) Determining structures of RNA aptamers and riboswitches by X-ray crystallography. Methods Mol Biol 535:135–163PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Reyes FE, Garst AD, Batey RT (2009) Strategies in RNA crystallography. Methods Enzymol 469:119–139PubMedCrossRefGoogle Scholar
  51. 51.
    Galsbøl FH, Simonsen K (1990) The preparation, separation and characterization of some ammine complexes or Iridium(III). Acta Chem Scand 44:796–801CrossRefGoogle Scholar
  52. 52.
    Golden BL (2007) Preparation and crystallization of RNA. Methods Mol Biol 363:239–257PubMedCrossRefGoogle Scholar
  53. 53.
    Golden BL, Kundrot CE (2003) RNA crystallization. J Struct Biol 142:98–107PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of Colorado at BoulderBoulderUSA
  2. 2.Department of Biochemistry and Molecular Genetics and Howard Hughes Medical InstituteUniversity of Colorado Denver School of MedicineAuroraUSA

Personalised recommendations