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High-resolution membrane protein structure by joint calculations with solid-state NMR and X-ray experimental data

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Abstract

X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) are the staple methods for revealing atomic structures of proteins. Since crystals of biomolecular assemblies and membrane proteins often diffract weakly and such large systems encroach upon the molecular tumbling limit of solution NMR, new methods are essential to extend structures of such systems to high resolution. Here we present a method that incorporates solid-state NMR restraints alongside of X-ray reflections to the conventional model building and refinement steps of structure calculations. Using the 3.7 Å crystal structure of the integral membrane protein complex DsbB-DsbA as a test case yielded a significantly improved backbone precision of 0.92 Å in the transmembrane region, a 58% enhancement from using X-ray reflections alone. Furthermore, addition of solid-state NMR restraints greatly improved the overall quality of the structure by promoting 22% of DsbB transmembrane residues into the most favored regions of Ramachandran space in comparison to the crystal structure. This method is widely applicable to any protein system where X-ray data are available, and is particularly useful for the study of weakly diffracting crystals.

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Acknowledgments

This research was supported by the National Institutes of Health (R01GM075937, S10RR025037, and R01GM073770 ARRA supplement to Chad M. Rienstra, and NRSA F32GM095344 to Anna E. Nesbitt), the Molecular Biophysics Training Grant (PHS 5 T32 GM008276) and Ullyot Fellowship to Lindsay J. Sperling, and the NIH Intramural Research Program of CIT to Charles D. Schwieters. The authors thank the School of Chemical Sciences NMR Facility at the University of Illinois at Urbana-Champaign for assistance with data acquisition and Mike Hallock for helpful assistance with structure calculations.

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Authors and Affiliations

  1. Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801, USA

    Ming Tang, Lindsay J. Sperling, Deborah A. Berthold, Anna E. Nesbitt, Andrew J. Nieuwkoop, Robert B. Gennis & Chad M. Rienstra

  2. Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, MD, 20892, USA

    Charles D. Schwieters

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  1. Ming Tang
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  2. Lindsay J. Sperling
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Correspondence to Chad M. Rienstra.

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10858_2011_9565_MOESM1_ESM.pdf

Supplementary material 1 Experimental procedures, 13C-13C correlation spectra of glycerol labeled DsbB samples and validations of structural calculations of DsbB-DsbA. (PDF 443 kb)

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Tang, M., Sperling, L.J., Berthold, D.A. et al. High-resolution membrane protein structure by joint calculations with solid-state NMR and X-ray experimental data. J Biomol NMR 51, 227–233 (2011). https://doi.org/10.1007/s10858-011-9565-6

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