Atomic resolution protein structure determination by three-dimensional transferred echo double resonance solid-state nuclear magneticresonance spectroscopy

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 - C 13 or 2 - C 13 -glycerol as the C 13 source. Quantitative dipolar trajectories are extracted from two-dimensional N 15 - C 13 planes, in which ∼ 750 cross peaks are resolved. The experimental data are fit to exact theoretical trajectories for spin clusters (consisting of one C 13 and several N 15 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.