title: NMR data collection and analysis protocol for high-throughput protein structure determination.
abstract: 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. Between 1 and 9 days of instrument time were invested per structure, which is less than approximately 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 approximately 20 kDa, while concurrently providing spectra that are highly amenable to fast and robust analysis.
authors: Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY 14260, USA.
authors: Gaohua
authors: G
authors: Liu
authors: Yang
authors: Y
authors: Shen
authors: Hanudatta S
authors: HS
authors: Atreya
authors: David
authors: D
authors: Parish
authors: Ying
authors: Y
authors: Shao
authors: Dinesh K
authors: DK
authors: Sukumaran
authors: Rong
authors: R
authors: Xiao
authors: Adelinda
authors: A
authors: Yee
authors: Alexander
authors: A
authors: Lemak
authors: Aneerban
authors: A
authors: Bhattacharya
authors: Thomas A
authors: TA
authors: Acton
authors: Cheryl H
authors: CH
authors: Arrowsmith
authors: Gaetano T
authors: GT
authors: Montelione
authors: Thomas
authors: T
authors: Szyperski
mesh_terms: D003625:Data Collection / Q000379:methods; D005583:Fourier Analysis; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins