TY - JOUR
T1 - Atomic resolution protein structure determination by three-dimensional transferred echo double resonance solid-state nuclear magnetic resonance spectroscopy
AU - Nieuwkoop, Andrew J.
AU - Wylie, Benjamin J.
AU - Franks, W. Trent
AU - Shah, Gautam J.
AU - Rienstra, Chad M.
N1 - Funding Information:
This work was supported by the National Institutes of Health Grant No. R01-GM073770. A.J.N. thanks the Molecular Biophysics Training Program at the University of Illinois for financial support.
PY - 2009
Y1 - 2009
N2 - We show that quantitative internuclear N 15 - C 13 distances can be obtained in sufficient quantity to determine a complete, high-resolution structure of a moderately sized protein by magic-angle spinning solid-state NMR spectroscopy. The three-dimensional ZF-TEDOR pulse sequence is employed in combination with sparse labeling of C 13 sites in the Β 1 domain of the immunoglobulin binding protein G (GB1), as obtained by bacterial expression with 1,3- C13 or 2- C 13-glycerol as the C13 source. Quantitative dipolar trajectories are extracted from two-dimensional N 15 - 13C planes, in which ∼750 cross peaks are resolved. The experimental data are fit to exact theoretical trajectories for spin clusters (consisting of one 13C and several 15N each), yielding quantitative precision as good as 0.1 Å for ∼350 sites, better than 0.3 Å for another 150, and ∼1.0 Å for 150 distances in the range of 5-8 Å. Along with isotropic chemical shift-based (TALOS) dihedral angle restraints, the distance restraints are incorporated into simulated annealing calculations to yield a highly precise structure (backbone RMSD of 0.25±0.09 Å), which also demonstrates excellent agreement with the most closely related crystal structure of GB1 (2QMT, bbRMSD 0.79±0.03 Å). Moreover, side chain heavy atoms are well restrained (0.76±0.06 Å total heavy atom RMSD). These results demonstrate for the first time that quantitative internuclear distances can be measured throughout an entire solid protein to yield an atomic-resolution structure.
AB - We show that quantitative internuclear N 15 - C 13 distances can be obtained in sufficient quantity to determine a complete, high-resolution structure of a moderately sized protein by magic-angle spinning solid-state NMR spectroscopy. The three-dimensional ZF-TEDOR pulse sequence is employed in combination with sparse labeling of C 13 sites in the Β 1 domain of the immunoglobulin binding protein G (GB1), as obtained by bacterial expression with 1,3- C13 or 2- C 13-glycerol as the C13 source. Quantitative dipolar trajectories are extracted from two-dimensional N 15 - 13C planes, in which ∼750 cross peaks are resolved. The experimental data are fit to exact theoretical trajectories for spin clusters (consisting of one 13C and several 15N each), yielding quantitative precision as good as 0.1 Å for ∼350 sites, better than 0.3 Å for another 150, and ∼1.0 Å for 150 distances in the range of 5-8 Å. Along with isotropic chemical shift-based (TALOS) dihedral angle restraints, the distance restraints are incorporated into simulated annealing calculations to yield a highly precise structure (backbone RMSD of 0.25±0.09 Å), which also demonstrates excellent agreement with the most closely related crystal structure of GB1 (2QMT, bbRMSD 0.79±0.03 Å). Moreover, side chain heavy atoms are well restrained (0.76±0.06 Å total heavy atom RMSD). These results demonstrate for the first time that quantitative internuclear distances can be measured throughout an entire solid protein to yield an atomic-resolution structure.
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U2 - 10.1063/1.3211103
DO - 10.1063/1.3211103
M3 - Article
C2 - 19739873
AN - SCOPUS:69949160677
SN - 0021-9606
VL - 131
JO - Journal of Chemical Physics
JF - Journal of Chemical Physics
IS - 9
M1 - 095101
ER -