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

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Nucleic Acid Crystallography

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1320))

Abstract

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.

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Colorado at Boulder, 596 UCB, Boulder, CO, 80309, USA

    Robert T. Batey

  2. Department of Biochemistry and Molecular Genetics and Howard Hughes Medical Institute, University of Colorado Denver School of Medicine, Mail Stop 8101, Research-1S, 12801 East 17th Ave., Room L18-9110, Aurora, CO, 80045, USA

    Jeffrey S. Kieft

Authors
  1. Robert T. Batey
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  2. Jeffrey S. Kieft
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Correspondence to Robert T. Batey or Jeffrey S. Kieft .

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Editors and Affiliations

  1. CNRS Inst de Biol Moléculaire et Cellulaire, Strasbourg, France

    Eric Ennifar

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Batey, R.T., Kieft, J.S. (2016). Soaking Hexammine Cations into RNA Crystals to Obtain Derivatives for Phasing Diffraction Data. In: Ennifar, E. (eds) Nucleic Acid Crystallography. Methods in Molecular Biology, vol 1320. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2763-0_14

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  • DOI: https://doi.org/10.1007/978-1-4939-2763-0_14

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2762-3

  • Online ISBN: 978-1-4939-2763-0

  • eBook Packages: Springer Protocols

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