Post-crystallization Improvement of RNA Crystal Diffraction Quality

  • Jinwei Zhang
  • Adrian R. Ferré-D’AmaréEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1316)


The crystallization and structural determination of large RNAs and their complexes remain major bottlenecks in the mechanistic analysis of cellular and viral RNAs. Here, we describe a protocol that combines post-crystallization dehydration and ion replacement that dramatically improved the diffraction quality of crystals of a large gene-regulatory tRNA–mRNA complex. Through this method, the resolution limit of X-ray data extended from 8.5 to 3.2 Å, enabling structure determination. Although this protocol was developed for a particular RNA complex, the general importance of solvent and counterions in nucleic acid structure may render it generally useful for crystallographic analysis of other RNAs.

Key words

X-ray crystallography Crystal dehydration Ion replacement Riboswitch T-box RNA tRNA 



We thank the staff at beamlines 5.0.1 and 5.0.2 of the ALS and ID-24-C and ID-24-E of APS, in particular, K. Perry and K.R. Rajashankar of the Northeastern Collaborative Access Team (NE-CAT) of the APS for support in data collection and processing, G. Piszczek (National Heart, Lung and Blood Institute, NHLBI), R. Levine and D.-Y. Lee (NHLBI) for assistance with biophysical and mass spectrometric characterization, and N. Baird, T. Hamma, C. Jones, M. Lau, A. Roll-Mecak, and K. Warner for discussions. This work is partly based on research conducted at the ALS on the Berkeley Center for Structural Biology beamlines and at the APS on the NE-CAT beamlines (supported by National Institute of General Medical Sciences grant P41GM103403). Use of ALS and APS was supported by the US Department of Energy. This work was supported in part by the intramural program of the NHLBI, NIH.


  1. 1.
    Cech TR, Steitz JA (2014) The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157:77–94CrossRefPubMedGoogle Scholar
  2. 2.
    Watts JM, Dang KK, Gorelick RJ, Leonard CW, Bess JW Jr, Swanstrom R, Burch CL, Weeks KM (2009) Architecture and secondary structure of an entire HIV-1 RNA genome. Nature 460:711–716CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Fang X, Wang J, O'Carroll IP, Mitchell M, Zuo X, Wang Y, Yu P, Liu Y, Rausch JW, Dyba MA, Kjems J, Schwieters CD, Seifert S, Winans RE, Watts NR, Stahl SJ, Wingfield PT, Byrd RA, Le Grice SF, Rein A, Wang YX (2013) An unusual topological structure of the HIV-1 Rev response element. Cell 155:594–605CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Cantara WA, Olson ED, Musier-Forsyth K (2014) Progress and outlook in structural biology of large viral RNAs. Virus Res 193:24–38CrossRefPubMedGoogle Scholar
  5. 5.
    Zhang J, Ferre-D’Amare AR (2014) New molecular engineering approaches for crystallographic studies of large RNAs. Curr Opin Struct Biol 26C:9–15CrossRefGoogle Scholar
  6. 6.
    Heras B, Martin JL (2005) Post-crystallization treatments for improving diffraction quality of protein crystals. Acta Crystallogr D Biol Crystallogr 61:1173–1180CrossRefPubMedGoogle Scholar
  7. 7.
    Russo Krauss I, Sica F, Mattia CA, Merlino A (2012) Increasing the X-ray diffraction power of protein crystals by dehydration: the case of bovine serum albumin and a survey of literature data. Int J Mol Sci 13:3782–3800CrossRefPubMedGoogle Scholar
  8. 8.
    Deng X, Davidson WS, Thompson TB (2012) Improving the diffraction of apoA-IV crystals through extreme dehydration. Acta Crystallogr Sect F Struct Biol Cryst Commun 68:105–110CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Awad W, Svensson Birkedal G, Thunnissen MM, Mani K, Logan DT (2013) Improvements in the order, isotropy and electron density of glypican-1 crystals by controlled dehydration. Acta Crystallogr D Biol Crystallogr 69:2524–2533CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Klein DJ, Ferré-D’Amaré AR (2009) Crystallization of the glmS ribozyme-riboswitch. Methods Mol Biol 540:129–139CrossRefPubMedGoogle Scholar
  11. 11.
    Klein DJ, Ferré-D’Amaré AR (2006) Structural basis of glmS ribozyme activation by glucosamine-6-phosphate. Science 313:1752–1756CrossRefPubMedGoogle Scholar
  12. 12.
    Zhang J, Ferré-D’Amaré AR (2013) Co-crystal structure of a T-box riboswitch stem I domain in complex with its cognate tRNA. Nature 500:363–366CrossRefPubMedGoogle Scholar
  13. 13.
    Zhang J, Ferré-D’Amaré AR (2014) Dramatic improvement of crystals of large RNAs by cation replacement and dehydration. Structure 22(9):1363–1371CrossRefPubMedGoogle Scholar
  14. 14.
    Draper DE (2004) A guide to ions and RNA structure. RNA 10:335–343CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Grundy FJ, Henkin TM (1993) tRNA as a positive regulator of transcription antitermination in B. subtilis. Cell 74:475–482CrossRefPubMedGoogle Scholar
  16. 16.
    Milligan JF, Uhlenbeck OC (1989) Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol 180:51–62CrossRefPubMedGoogle Scholar
  17. 17.
    Zhang J, Ferre-D'Amare AR (2014) Direct evaluation of tRNA aminoacylation status by the T-Box riboswitch using tRNA-mRNA stacking and steric readout. Mol Cell 55:148–155CrossRefPubMedGoogle Scholar
  18. 18.
    Ferré-D'Amaré AR (2010) Use of the spliceosomal protein U1A to facilitate crystallization and structure determination of complex RNAs. Methods 52:159–167CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Shechner DM, Grant RA, Bagby SC, Koldobskaya Y, Piccirilli JA, Bartel DP (2009) Crystal structure of the catalytic core of an RNA-polymerase ribozyme. Science 326:1271–1275CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Koldobskaya Y, Duguid EM, Shechner DM, Suslov NB, Ye J, Sidhu SS, Bartel DP, Koide S, Kossiakoff AA, Piccirilli JA (2011) A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination. Nat Struct Mol Biol 18:100–106CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Klein DJ, Schmeing TM, Moore PB, Steitz TA (2001) The kink-turn: a new RNA secondary structure motif. EMBO J 20:4214–4221CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Winkler WC, Grundy FJ, Murphy BA, Henkin TM (2001) The GA motif: an RNA element common to bacterial antitermination systems, rRNA, and eukaryotic RNAs. RNA 7:1165–1172CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Daldrop P, Lilley DM (2013) The plasticity of a structural motif in RNA: structural polymorphism of a kink turn as a function of its environment. RNA 19:357–364CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Lilley DM (2012) The structure and folding of kink turns in RNA. Wiley Interdiscip Rev RNA 3:797–805CrossRefPubMedGoogle Scholar
  25. 25.
    Hamma T, Ferré-D'Amaré AR (2004) Structure of protein L7Ae bound to a K-turn derived from an archaeal box H/ACA sRNA at 1.8 Å resolution. Structure 12:893–903CrossRefPubMedGoogle Scholar
  26. 26.
    Baird NJ, Zhang J, Hamma T, Ferré-D'Amaré AR (2012) YbxF and YlxQ are bacterial homologs of L7Ae and bind K-turns but not K-loops. RNA 18:759–770CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    Biertümpfel C, Basquin J, Suck D, Sauter C (2002) Crystallization of biological macromolecules using agarose gel. Acta Crystallogr D Biol Crystallogr 58:1657–1659CrossRefPubMedGoogle Scholar
  28. 28.
    Lorber B, Sauter C, Theobald-Dietrich A, Moreno A, Schellenberger P, Robert MC, Capelle B, Sanglier S, Potier N, Giege R (2009) Crystal growth of proteins, nucleic acids, and viruses in gels. Prog Biophys Mol Biol 101:13–25CrossRefPubMedGoogle Scholar
  29. 29.
    Hofer TS, Randolf BR, Rode BM (2006) Sr(II) in water: a labile hydrate with a highly mobile structure. J Phys Chem B 110:20409–20417CrossRefPubMedGoogle Scholar
  30. 30.
    Mueller U, Schubel H, Sprinzl M, Heinemann U (1999) Crystal structure of acceptor stem of tRNA(Ala) from Escherichia coli shows unique G.U wobble base pair at 1.16 Å resolution. RNA 5:670–677CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.National Heart, Lung and Blood InstituteBethesdaUSA

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