TY - JOUR
T1 - Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein
AU - Lee, Jeffrey E.
AU - Fusco, Marnie L.
AU - Abelson, Dafna M.
AU - Hessell, Ann J.
AU - Burton, Dennis R.
AU - Saphire, Erica Ollmann
N1 - Copyright:
Copyright 2010 Elsevier B.V., All rights reserved.
PY - 2009
Y1 - 2009
N2 - 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 glyco-protein 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.
AB - 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 glyco-protein 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.
KW - Antibody complexes
KW - Deglycosylation
KW - Difficult structures
KW - Glycoproteins
KW - Human proteins
KW - Model building
KW - Structure determination
KW - Tactics to improve diffraction
KW - Techniques for phase determination
KW - Viral proteins
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U2 - 10.1107/S0907444909032314
DO - 10.1107/S0907444909032314
M3 - Article
C2 - 19923712
AN - SCOPUS:73349143321
VL - 65
SP - 1162
EP - 1180
JO - Acta Crystallographica Section D: Structural Biology
JF - Acta Crystallographica Section D: Structural Biology
SN - 0907-4449
IS - 11
ER -