TY - JOUR
T1 - NMR data collection and analysis protocol for high-throughput protein structure determination
AU - Liu, Gaohua
AU - Shen, Yang
AU - Atreya, Hanudatta S.
AU - Parish, David
AU - Shao, Ying
AU - Sukumaran, Dinesh K.
AU - Xiao, Rong
AU - Yee, Adelinda
AU - Lemak, Alexander
AU - Bhattacharya, Aneerban
AU - Acton, Thomas A.
AU - Arrowsmith, Cheryl H.
AU - Montelione, Gaetano T.
AU - Szyperski, Thomas
PY - 2005/7/26
Y1 - 2005/7/26
N2 - 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.
AB - 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.
KW - G-matrix Fourier transform projection NMR
KW - NMR structure determination
KW - Structural genomics
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U2 - 10.1073/pnas.0504338102
DO - 10.1073/pnas.0504338102
M3 - Article
C2 - 16027363
AN - SCOPUS:23044509110
SN - 0027-8424
VL - 102
SP - 10487
EP - 10492
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 30
ER -