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Microgram-scale protein structure determination by NMR

Nature Methods volume 4, pages 491–493 (2007)Cite this article

Abstract

Using conventional triple-resonance nuclear magnetic resonance (NMR) experiments with a 1 mm triple-resonance microcoil NMR probe, we determined near complete resonance assignments and three-dimensional (3D) structure of the 68-residue Methanosarcina mazei TRAM protein using only 72 μg (6 μl, 1.4 mM) of protein. This first example of a complete solution NMR structure determined using microgram quantities of protein demonstrates the utility of microcoil-probe NMR technologies for protein samples that can be produced in only limited quantities.

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Figure 1: Microprobe HSQC spectrum.
Figure 2: Comparison of NOESY spectral quality.
Figure 3: Assessment of structural accuracy.

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Acknowledgements

We thank T. Acton for helpful discussions. This work was supported by a grant from the Protein Structure Initiative of the US National Institutes of Health (U54 GM074958).

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Author notes

  1. James M Aramini and Paolo Rossi: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, and Northeast Structural Genomics Consortium (NESG), Rutgers University, Piscataway, 08854, New Jersey, USA

    James M Aramini, Paolo Rossi, Rong Xiao & Gaetano T Montelione

  2. Bruker BioSpin Corporation, 15 Fortune Drive, Manning Park, Billerica, 01821, Massachusetts, USA

    Clemens Anklin

  3. Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Piscataway, 08854, New Jersey, USA

    Gaetano T Montelione

Authors
  1. James M Aramini
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  2. Paolo Rossi
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  3. Clemens Anklin
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  4. Rong Xiao
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  5. Gaetano T Montelione
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Corresponding author

Correspondence to Gaetano T Montelione.

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Competing interests

C.A. is employed by the manufacturer of the NMR equipment used.

Supplementary information

Supplementary Fig. 1

Bruker 1 mm MicroProbe performance with NESG targets of increasing molecular weight, evaluated by 2D 1H-15N HSQC. (PDF 108 kb)

Supplementary Fig. 2

Connectivity map for Q8PX65 from Methanosarcina mazei based on triple-resonance data obtained using Bruker 1 mm MicroProbes. (PDF 184 kb)

Supplementary Fig. 3

Molecular weight and concentration distribution of NESG protein structures solved by NMR. (PDF 105 kb)

Supplementary Table 1

Comparison of mass-based sensitivity for various Bruker probeheads. (PDF 23 kb)

Supplementary Table 2

Complete NMR data and structural statistics for M. mazei Q8PX65. (PDF 93 kb)

Supplementary Methods (PDF 107 kb)

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Aramini, J., Rossi, P., Anklin, C. et al. Microgram-scale protein structure determination by NMR. Nat Methods 4, 491–493 (2007). https://doi.org/10.1038/nmeth1051

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