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首页> 外文期刊>Acta crystallographica.Section D. Biological crystallography >Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein
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Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein

机译：在决定使用技术和策略三聚物的结构ebolavirus糖蛋白

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摘要

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 fi-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.
机译：的三聚物的membrane-anchored ebolavirus信封负责病毒糖蛋白(GP)附件、融合和条目。结构对于理解是很重要的ebolavirus进入和发展的医疗干预措施。病毒糖蛋白,特别是那些在他们亚稳十二低聚物的状态,可以难以实现的挑战生产、纯化、结晶和衍射中固有的糖基化的这些类型的灵活的本质蛋白质。在复杂的三聚物的十二构象与人类抗体来自的幸存者1995年的Kikwit疫情已经被确定[李et al。(2008)、自然(伦敦),454177 - 182年)。策略用于克服一系列的技术路障在结晶和定相描述。胚胎肾293 t细胞,这允许快速筛选的结构和表达的蛋白质在毫克量。抗体片段(Fab)促进了结晶和一系列deglycosylation策略,包括糖突变体酶deglycosylation,研究进展和表达式多糖合成代谢通路抑制剂试图提高弱衍射糖蛋白晶体。为分子置换的搜索模型通过确定的结构改进非复杂工厂。阶段和硒异常信号,跟踪通过NCS-averaged密度修正,导致一个明确的可翻译的电子密度图。建筑是辅助使用fi-value-sharpened电子密度和地图适当的序列证实了注册建筑使用N-linked交替序列多糖作为主持人和secondary-structural网站预测。

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