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

Gaohua Liu, Yang Shen, Hanudatta S. Atreya, David Parish, Ying Shao, Dinesh K. Sukumaran, Rong Xiao, Adelinda Yee, Alexander Lemak, Aneerban Bhattacharya, Thomas A. Acton, Cheryl H. Arrowsmith, Gaetano T. Montelione, Thomas Szyperski

Research output: Contribution to journalArticlepeer-review

101 Scopus citations

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,13Caliphatic,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.

Original languageEnglish (US)
Pages (from-to)10487-10492
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number30
DOIs
StatePublished - Jul 26 2005

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • G-matrix Fourier transform projection NMR
  • NMR structure determination
  • Structural genomics

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