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Major groove width variations in RNA structures determined by NMR and impact of 13C residual chemical shift anisotropy and 1H–13C residual dipolar coupling on refinement

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Abstract

Ribonucleic acid structure determination by NMR spectroscopy relies primarily on local structural restraints provided by 1H– 1H NOEs and J-couplings. When employed loosely, these restraints are broadly compatible with A- and B-like helical geometries and give rise to calculated structures that are highly sensitive to the force fields employed during refinement. A survey of recently reported NMR structures reveals significant variations in helical parameters, particularly the major groove width. Although helical parameters observed in high-resolution X-ray crystal structures of isolated A-form RNA helices are sensitive to crystal packing effects, variations among the published X-ray structures are significantly smaller than those observed in NMR structures. Here we show that restraints derived from aromatic 1H– 13C residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs) can overcome NMR restraint and force field deficiencies and afford structures with helical properties similar to those observed in high-resolution X-ray structures.

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Acknowledgments

Support from the NIH (GM42561 to M.F.S., GM45811 to D.A.C.) and the Intramural Research Program of the NIDDK, NIH (DK029051-03 to A.B.) is gratefully acknowledged. B.K., P.S., S.B. and R.S. are UMBC Meyerhoff Scholars and were supported by an HHMI undergraduate education grant.

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  1. Blanton S. Tolbert

    Present address: Department of Chemistry and Biochemistry, Miami University of Ohio, Oxford, OH, 45056, USA

Authors and Affiliations

  1. Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA

    Blanton S. Tolbert, Yasuyuki Miyazaki, Shawn Barton, Benyam Kinde, Patrice Starck, Rashmi Singh & Michael F. Summers

  2. Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, MD, 208992, USA

    Ad Bax

  3. Department of Chemistry & Chemical Biology and BioMaPS Institute, Rutgers University, 610 Taylor Road, Piscataway, NJ, 08854-8087, USA

    David A. Case

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  1. Blanton S. Tolbert
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Correspondence to David A. Case or Michael F. Summers.

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10858_2010_9424_MOESM1_ESM.pdf

A suite of scripts for generating restraints to maintain aromatic base planarity and ideal bond angles, generating Amber compatible RCSA and RDC restraints, performing MPI-based Amber structure calculations, facilitating semi-automated structure analysis with Pymol and X3DNA, and Bruker-compatible IMC pulse sequence, parameter, and sample data files, are available for download at: www.hhmi.umbc.edu/downloads. Coordinates for the final, refined [DIS]2 structures, and associated experimental RDC and RCSA values, have been deposited (PDB 2kyd (rscb 101717) and BMRB 16980, respectively). Details on the implementation of RCSAs in Amber, and raw NMR frequencies and calculated RDC, RCSA and PRCSA values for [DIS]2. (PDF 311 kb)

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Tolbert, B.S., Miyazaki, Y., Barton, S. et al. Major groove width variations in RNA structures determined by NMR and impact of 13C residual chemical shift anisotropy and 1H–13C residual dipolar coupling on refinement. J Biomol NMR 47, 205–219 (2010). https://doi.org/10.1007/s10858-010-9424-x

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