In this paper, a three-dimensional (3D) NMR-based approach for the determination of the fold of moderately sized proteins by solid-state magic-angle spinning (MAS) NMR is presented and applied to the alpha-spectrin SH3 domain. This methodology includes the measurement of multiple C-13-C-13 distance restraints on biosynthetically site-directed C-13-enriched samples, obtained by growing bacteria on [2-C-13]glycerol and [1,3-C-13]glycerol. 3D N-15-C-13-C-13 dipolar correlation experiments were applied to resolve overlap of signals, in particular in the region where backbone carbon-carbon correlations of the C-alpha-Calpha, CO-CO, C-alpha-CO, and CO-C-alpha type appear. Additional restraints for confining the structure were obtained from phi and psi backbone torsion angles of 29 residues derived from C-alpha, C-beta, CO, NH, and Ha chemical shifts. Using both distance and angular restraints, a refined structure was calculated with a backbone root-mean-square deviation of 0.7 Angstrom with respect to the average structure.
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