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PDBsum entry 1kme
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Membrane protein
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PDB id
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1kme
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure.
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Authors
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S.Faham,
J.U.Bowie.
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Ref.
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J Mol Biol, 2002,
316,
1-6.
[DOI no: ]
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PubMed id
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Abstract
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Obtaining crystals of membrane proteins that diffract to high resolution remains
a major stumbling block in structure determination. Here we present a new method
for crystallizing membrane proteins from a bicelle forming lipid/detergent
mixture. The method is flexible and simple to use. As a test case,
bacteriorhodopsin (bR) from Halobacterium salinarum was crystallized from a
bicellar solution, yielding a new bR crystal form. The crystals belong to space
group P2(1) with unit cell dimensions of a=45.0 A, b=108.9 A, c=55.9 A,
beta=113.58 degrees and a dimeric asymmetric unit. The structure was solved by
molecular replacement and refined at 2.0 A resolution. In all previous bR
structures the protein is organized as a parallel trimer, but in the crystals
grown from bicelles, the individual bR subunits are arranged in an antiparallel
fashion.
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Figure 1.
Figure 1. Bicelle crystallization method. (a) Outline of
the method. (b) Crystals of bR grown from bicelle forming
lipid/detergent mixture. Purple membrane was purified as
described by Oesterhelt & Steckenius.[29] Purple membrane was
suspended in water to a bR concentration of  vert,
similar 10 mg/ml and was mixed in a 4:1 ratio with a 40% (3:1)
DMPC/Chapso bicellar solution, making a vert,
similar 8.0 mg/ml bR/8% bicelles mixture. Reconstitution and
homogenization of the protein into the bicelles was achieved
simply by repeated pipeting of the solution. Crystals were grown
using the hanging, or sitting drop method and all solutions were
kept on ice prior to mixing to keep the bicelle mixtures fluid.
The crystallization drops contained 6 µl of
protein/bicelle solution with 2.5 µl of well solution and
1 µl of 2.5% b-octylglucoside (OG) solution. The well
solution contained 3.2 M NaPO[4] (pH 3.5). The crystal trays
were then placed in a 37°C incubator to allow the bicelles
to gel. Diamond-shaped crystals grew within a few days to two
weeks. The addition of OG was not required for crystal growth;
however, larger crystals were obtained in its presence.
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Figure 3.
Figure 3. The packing interfaces in the bicelle crystals.
(a) Top view of a layer. (b) Side view of a layer. Helices A and
B (red) and helices C and D (blue), provide the main
contributions to interface I. Helices E and F (green) provide
the main contributions to interface II. Figure 2 and Figure 3
were prepared using the program MOLSCRIPT. [30]
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2002,
316,
1-6)
copyright 2002.
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