Journal of Biomolecular NMR

, Volume 38, Issue 4, pp 341–351 | Cite as

HIFI-C: a robust and fast method for determining NMR couplings from adaptive 3D to 2D projections



We describe a novel method for the robust, rapid, and reliable determination of J couplings in multi-dimensional NMR coupling data, including small couplings from larger proteins. The method, “High-resolution Iterative Frequency Identification of Couplings” (HIFI-C) is an extension of the adaptive and intelligent data collection approach introduced earlier in HIFI-NMR. HIFI-C collects one or more optimally tilted two-dimensional (2D) planes of a 3D experiment, identifies peaks, and determines couplings with high resolution and precision. The HIFI-C approach, demonstrated here for the 3D quantitative J method, offers vital features that advance the goal of rapid and robust collection of NMR coupling data. (1) Tilted plane residual dipolar couplings (RDC) data are collected adaptively in order to offer an intelligent trade off between data collection time and accuracy. (2) Data from independent planes can provide a statistical measure of reliability for each measured coupling. (3) Fast data collection enables measurements in cases where sample stability is a limiting factor (for example in the presence of an orienting medium required for residual dipolar coupling measurements). (4) For samples that are stable, or in experiments involving relatively stronger couplings, robust data collection enables more reliable determinations of couplings in shorter time, particularly for larger biomolecules. As a proof of principle, we have applied the HIFI-C approach to the 3D quantitative J experiment to determine N-C′ RDC values for three proteins ranging from 56 to 159 residues (including a homodimer with 111 residues in each subunit). A number of factors influence the robustness and speed of data collection. These factors include the size of the protein, the experimental set up, and the coupling being measured, among others. To exhibit a lower bound on robustness and the potential for time saving, the measurement of dipolar couplings for the N-C′ vector represents a realistic “worst case analysis”. These couplings are among the smallest currently measured, and their determination in both isotropic and anisotropic media demands the highest measurement precision. The new approach yielded excellent quantitative agreement with values determined independently by the conventional 3D quantitative J NMR method (in cases where sample stability in oriented media permitted these measurements) but with a factor of 2–5 in time savings. The statistical measure of reliability, measuring the quality of each RDC value, offers valuable adjunct information even in cases where modest time savings may be realized.


HIFI-C High-resolution Iterative Frequency Identification of Couplings Fast NMR data collection NMR coupling measurements RDC Residual dipolar couplings Adaptive reduced-dimensionality 



We thank Ad Bax and Sam Butcher for allowing us to use their protein samples GB3 and Prp24-12, respectively, in this study. This research was carried out in the National Magnetic Resonance Facility at Madison, which is supported by Biomedical Research Technology Program, National Center for Research Resources, through NIH grant P41 RR02301 and utilized equipment funded by the University of Wisconsin, the NSF Academic Infrastructure Program (BIR-9214394), the NIH Shared Instrumentation Program (RR02781, RR08438), the NIH Research Collaborations to Provide 900 MHz NMR Spectroscopy (GM66326), the NSF Biological Instrumentation Program (DMB-8415048), and the U.S. Department of Agriculture. A.B. received partial support from the National Institute of General Medical Science’s Protein Structure Initiative through NIH grant 1 P50 GM64598, which supports the Center for Eukaryotic Structural Genomics. H.E. was supported in part by the National Library of Medicine under grant 1K22LM8992.

Supplementary material

10858_2007_9173_MOESM1_ESM.doc (920 kb)
One figure with correlation plots between experimental (obtained by 3D and HIFI-C methods) and GB3 structure fitted RDCs, one table containing the statistical analysis of isotropic couplings extracted from three different HIFI-C tilt angles for protein At5g22580.1, one table containing three outliers in the comparison of RDCs measured by the HIFI-C and 3D quantitative J methods for protein GB3, one table containing the analysis of the outliers in the comparison between NC′ RDC values measured by the HIFI-C and 3D quantitative J methods for the 25 kDa homodimeric At5g22580.1 protein, one figure with the At5g22580.1 correlation between RDCs extracted from the 3D experiment versus those from HIFI-C at three tilted plane angles (28°, 57° and 77°), one figure with the At5g22580.1 correlation between the NC′ couplings from each pair of “+/−” HIFI-C tilt planes (for three tilt angles: 28°, 57° and 77°) in isotropic and aligned state, one figure with comparisons of the At5g22580.1 NC′ couplings between different HIFI tilt planes in isotropic state and aligned states, two plots comparing 1D traces from the 3D experiment with those from the 2D HIFI-C planes for GB3 protein in isotropic and aligned states, respectively, and two plots comparing the 1D traces from the 3D experiment with those from the 2D HIFI-C planes for the At5g22580.1 protein, in isotropic and aligned states, respectively. ESM1 (DOC 921 kb)


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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Gabriel Cornilescu
    • 1
  • Arash Bahrami
    • 1
    • 2
    • 3
  • Marco Tonelli
    • 1
  • John L. Markley
    • 1
    • 2
    • 3
  • Hamid R. Eghbalnia
    • 1
    • 2
    • 3
    • 4
  1. 1.National Magnetic Resonance Facility at MadisonUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Center for Eukaryotic Structural GenomicsUniversity of Wisconsin-MadisonMadisonUSA
  3. 3.Department of Biochemistry, Graduate Program in BiophysicsUniversity of Wisconsin-MadisonMadisonUSA
  4. 4.Department of MathematicsUniversity of Wisconsin-MadisonMadisonUSA

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