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. 2011 Nov;51(3):227-33.
doi: 10.1007/s10858-011-9565-6. Epub 2011 Sep 22.

High-resolution Membrane Protein Structure by Joint Calculations With Solid-State NMR and X-ray Experimental Data

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Free PMC article

High-resolution Membrane Protein Structure by Joint Calculations With Solid-State NMR and X-ray Experimental Data

Ming Tang et al. J Biomol NMR. .
Free PMC article

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.

Figures

Figure 1
Figure 1
Overlay of 10 lowest-energy structures of DsbB-DsbA, calculated against (A) only X-ray reflections and (B) X-ray reflections and SSNMR restraints. The colors represent the secondary structure elements (magenta:α-helix, cyan and white: coil and turn, yellow:β-strand). The grey band indicates the membrane. The bbRMSD of the transmembrane regions has improved 58% with the joint calculation.
Figure 2
Figure 2
Comparison of chemical shifts of DsbB in solution (C44S, C104S) (Zhou et al. 2008) and in solid state (C41S). (A) 13CO chemical shifts, RMSD = 0.56 ppm; (B) 13Cα chemical shifts, RMSD = 0.70 ppm; (C) 13Cβ chemical shifts, RMSD = 0.95 ppm; (D) 15N chemical shifts, RMSD = 1.30 ppm. Outliers are marked.
Figure 3
Figure 3
Comparison of 2D expansions of the13C-13C correlation spectra of (A) U-DsbB, (B) U-DsbB/DsbA, (C) U-DsbA and (D) DsbB/U-DsbA. The spectra were collected at 750 MHz (1H frequency) with a spinning speed of 12.5 kHz and 25 ms DARR mixing.

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