A standardized protocol enabling rapid NMR data collection for high-quality protein structure determination is presented that allows one to capitalize on high spectrometer sensitivity: a set of five G-matrix Fourier transform NMR experiments for resonance assignment based on highly resolved 4D and 5D spectral information is acquired in conjunction with a single simultaneous 3D 15N,13C(aliphatic),13C(aromatic)-resolved [1H,1H]-NOESY spectrum providing 1H-1H upper distance limit constraints. The protocol was integrated with methodology for semiautomated data analysis and used to solve eight NMR protein structures of the Northeast Structural Genomics Consortium pipeline. The molecular masses of the hypothetical target proteins ranged from 9 to 20 kDa with an average of approximately 14 kDa.
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