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Title
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Cloning, purification, crystallization, and preliminary X-ray diffraction analysis of cystathionine gamma-synthase from E. coli.
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Authors
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M.C.Wahl,
R.Huber,
L.Prade,
S.Marinkovic,
A.Messerschmidt,
T.Clausen.
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Ref.
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Febs Lett, 1997,
414,
492-496.
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PubMed id
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Abstract
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The Escherichia coli metB gene has been PCR-extracted from genomic DNA and
placed under the control of a tac and a T7 promoter in plasmids pCYB1 and
pET22b(+), respectively, to produce overexpressing bacterial strains for the
gene product, cystathionine gamma-synthase. Efficient purification procedures
have been developed for a C-terminally intein-tagged version and the wild-type
target protein, yielding the product in a quantity and homogeneity amenable to
high-resolution single-crystal X-ray analysis. Crystals have been obtained in
space group P1 with unit cell constants a=82.2 A, b=84.2 A, c=116.2 A,
alpha=107.0 degrees, beta=96.3 degrees, gamma=108.0 degrees, suggesting eight
monomers per asymmetric unit (V[M]=2.23 A3/Da). Crystals diffract to beyond 2.6
A resolution and a data set complete to 2.8 A resolution has been collected
using a rotating anode X-ray source. A cryogenic buffer system has been
developed to allow synchrotron data collection. Patterson self rotation searches
reveal the presence of two independent tetramers with local 222 symmetry in an
asymmetric unit. The crystallographic results corroborate and extend previous
solution studies regarding the quaternary organization of the enzyme.
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