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PDBsum entry 1xn8
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Structural genomics, unknown function
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PDB id
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1xn8
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References listed in PDB file
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Key reference
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Title
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Nmr data collection and analysis protocol for high-Throughput protein structure determination.
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Authors
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G.Liu,
Y.Shen,
H.S.Atreya,
D.Parish,
Y.Shao,
D.K.Sukumaran,
R.Xiao,
A.Yee,
A.Lemak,
A.Bhattacharya,
T.A.Acton,
C.H.Arrowsmith,
G.T.Montelione,
T.Szyperski.
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Ref.
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Proc Natl Acad Sci U S A, 2005,
102,
10487-10492.
[DOI no: ]
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PubMed id
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Abstract
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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.
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Figure 1.
Fig. 1. Composite plot of 2D [^15N,^1H] HSQC spectra
recorded at 750 MHz for target proteins. Gene name, NESG target
ID, and number of amino acid residues (including tags) are
indicated in the top left of each plot. At the lower right, the
fraction of the peaks registered in these spectra is indicated
for which sequence specific resonance assignments were obtained.
For the highly  -helical protein yqbG
(Fig. 2), the central region is expanded in an Inset.
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Figure 2.
Fig. 2. High-quality NMR solution structures of target
proteins are displayed in the order of Table 1. For each
structure, a ribbon drawing is shown on the left. -Helices
are enumerated with roman numerals, and  -strands are indicated
with letters (for sequence locations of the regular secondary
structure elements, see footnote of Table 1). The N and C
termini of the polypeptide chains are labeled with N and C. On
the right, a "sausage" representation of the backboneis shown
for which a spline function was drawn through the C^ positions and where the
thickness of the cylindrical rod is proportional to the mean of
the global displacements of the 20 DYANA conformers calculated
after superposition of the backbone heavy atoms N, C^ , and C'
of the regular secondary structure elements for minimal rmsd.
Hence, the thickness reflects the precision achieved for the
determination of the polypeptide backbone conformation. A
superposition of the best-defined side chains having the lowest
global displacement for the side-chain heavy atoms also are
shown (best third of all residues; for residue numbers, see
footnote of Table 1) to indicate precision of the determination
of side-chain conformations. Helices are shown in red, the -stands
are depicted in cyan, other polypeptide segments are displayed
in gray, and the side chains of the molecular core are shown in
blue. The figure was generated by using the program MOLMOL (37).
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