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PDBsum entry 2j4y
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Signaling protein
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PDB id
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2j4y
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References listed in PDB file
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Key reference
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Title
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Crystal structure of a thermally stable rhodopsin mutant.
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Authors
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J.Standfuss,
G.Xie,
P.C.Edwards,
M.Burghammer,
D.D.Oprian,
G.F.Schertler.
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Ref.
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J Mol Biol, 2007,
372,
1179-1188.
[DOI no: ]
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PubMed id
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Abstract
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We determined the structure of the rhodopsin mutant N2C/D282C expressed in
mammalian cells; the first structure of a recombinantly produced G
protein-coupled receptor (GPCR). The mutant was designed to form a disulfide
bond between the N terminus and loop E3, which allows handling of opsin in
detergent solution and increases thermal stability of rhodopsin by 10 deg.C. It
allowed us to crystallize a fully deglycosylated rhodopsin (N2C/N15D/D282C). N15
mutations are normally misfolding and cause retinitis pigmentosa in humans.
Microcrystallographic techniques and a 5 mum X-ray beam were used to collect
data along a single needle measuring 5 mumx5 mumx90 mum. The disulfide
introduces only minor changes but fixes the N-terminal cap over the beta-sheet
lid covering the ligand-binding site, a likely explanation for the increased
stability. This work allows structural investigation of rhodopsin mutants and
shows the problems encountered during structure determination of GPCRs and other
mammalian membrane proteins.
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Figure 4.
Figure 4. Electron density maps of N2C/D282C rhodopsin. (a)
Retinal omit electron density map (0.75 σ) obtained after
molecular replacement and density modification. The retinal
(red) was not included but appears clearly in the electron
density. (b) Disulfide bond and N15 glycosylation. The
σA-weighted 2F[o] – F[c] with electron density contoured at
0.9σ. The disulfide bond between the mutated residues C2 and
C282 is well resolved, as is the first residue of the
N-glycosylation linked to N15. No density is visible adjacent to
the mutated residue 2, which would have been glycosylated in
wild-type rhodopsin.
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Figure 6.
Figure 6. The N-terminal cap domain. Contacts between the
N-terminal cap (blue) and the rest of the receptor (cyan). Atoms
of the receptor with a minimum distance of 5 Å to residues
1–33 of the N terminus are colored beige. The covalently bound
retinal (red) is separated from the N-terminal cap by the lid
formed from the E2 loop. The designed disulfide between C2 and
C282 connecting the N-terminal cap with loop E3 is shown as
yellow sticks. The molecule is viewed with the extracellular
site facing up.
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The above figures are
reprinted
from an Open Access publication published by Elsevier:
J Mol Biol
(2007,
372,
1179-1188)
copyright 2007.
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