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PDBsum entry 3inu
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Immune system
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PDB id
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3inu
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References listed in PDB file
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Key reference
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Title
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Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein.
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Authors
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J.E.Lee,
M.L.Fusco,
D.M.Abelson,
A.J.Hessell,
D.R.Burton,
E.O.Saphire.
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Ref.
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Acta Crystallogr D Biol Crystallogr, 2009,
65,
1162-1180.
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PubMed id
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Abstract
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The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is
responsible for viral attachment, fusion and entry. Knowledge of its structure
is important both for understanding ebolavirus entry and for the development of
medical interventions. Crystal structures of viral glycoproteins, especially
those in their metastable prefusion oligomeric states, can be difficult to
achieve given the challenges in production, purification, crystallization and
diffraction that are inherent in the heavily glycosylated flexible nature of
these types of proteins. The crystal structure of ebolavirus GP in its trimeric
prefusion conformation in complex with a human antibody derived from a survivor
of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008), Nature
(London), 454, 177-182]. Here, the techniques, tactics and strategies used to
overcome a series of technical roadblocks in crystallization and phasing are
described. Glycoproteins were produced in human embryonic kidney 293T cells,
which allowed rapid screening of constructs and expression of protein in
milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted
crystallization and a series of deglycosylation strategies, including sugar
mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic
pathway inhibitors, were attempted to improve the weakly diffracting
glycoprotein crystals. The signal-to-noise ratio of the search model for
molecular replacement was improved by determining the structure of the
uncomplexed Fab. Phase combination with Fab model phases and a selenium
anomalous signal, followed by NCS-averaged density modification, resulted in a
clear interpretable electron-density map. Model building was assisted by the use
of B-value-sharpened electron-density maps and the proper sequence register was
confirmed by building alternate sequences using N-linked glycan sites as anchors
and secondary-structural predictions.
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