NMR data collection and analysis protocol for high-throughput protein structure determination

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 ≈14 kDa. Between 1 and 9 days of instrument time were invested per structure, which is less than ≈10-25% of the measurement time routinely required to date with conventional approaches. The protocol presented here effectively removes data collection as a bottleneck for high-throughput solution structure determination of proteins up to at least ≈20 kDa, while concurrently providing spectra that are highly amenable to fast and robust analysis.