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

We show that quantitative internuclear (15)N-(13)C 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 (13)C sites in the beta1 domain of the immunoglobulin binding protein G (GB1), as obtained by bacterial expression with 1,3-(13)C or 2-(13)C-glycerol as the (13)C source. Quantitative dipolar trajectories are extracted from two-dimensional (15)N-(13)C planes, in which approximately 750 cross peaks are resolved. The experimental data are fit to exact theoretical trajectories for spin clusters (consisting of one (13)C and several (15)N each), yielding quantitative precision as good as 0.1 A for approximately 350 sites, better than 0.3 A for another 150, and approximately 1.0 A for 150 distances in the range of 5-8 A. 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 A), which also demonstrates excellent agreement with the most closely related crystal structure of GB1 (2QMT, bbRMSD 0.79+/-0.03 A). Moreover, side chain heavy atoms are well restrained (0.76+/-0.06 A 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.