TY - JOUR
T1 - High-resolution membrane protein structure by joint calculations with solid-state NMR and X-ray experimental data
AU - Tang, Ming
AU - Sperling, Lindsay J.
AU - Berthold, Deborah A.
AU - Schwieters, Charles D.
AU - Nesbitt, Anna E.
AU - Nieuwkoop, Andrew J.
AU - Gennis, Robert B.
AU - Rienstra, Chad M.
N1 - Funding Information:
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.
PY - 2011/11
Y1 - 2011/11
N2 - 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.
AB - 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.
KW - High resolution
KW - Joint calculation
KW - Membrane protein
KW - Solid-state NMR
KW - X-ray reflections
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U2 - 10.1007/s10858-011-9565-6
DO - 10.1007/s10858-011-9565-6
M3 - Article
C2 - 21938394
AN - SCOPUS:80755190085
SN - 0925-2738
VL - 51
SP - 227
EP - 233
JO - Journal of Biomolecular NMR
JF - Journal of Biomolecular NMR
IS - 3
ER -