Lee Jeffrey E, Fusco Marnie L, Abelson Dafna M, Hessell Ann J, Burton Dennis R, Saphire Erica Ollmann
Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA.
Acta Crystallogr D Biol Crystallogr. 2009 Nov;65(Pt 11):1162-80. doi: 10.1107/S0907444909032314. Epub 2009 Oct 22.
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.
三聚体膜锚定埃博拉病毒包膜糖蛋白（GP）负责病毒的附着、融合和进入。了解其结构对于理解埃博拉病毒的进入过程以及开发医学干预措施都很重要。鉴于这些高度糖基化的柔性蛋白质在生产、纯化、结晶和衍射方面存在固有挑战，获得病毒糖蛋白的晶体结构，尤其是其亚稳态预融合寡聚状态下的结构可能很困难。现已确定埃博拉病毒GP三聚体预融合构象与源自1995年基奎特疫情幸存者的人源抗体形成的复合物的晶体结构[Lee等人（2008年），《自然》（伦敦），454, 177 - 182]。本文描述了用于克服结晶和相位确定过程中一系列技术障碍所采用的技术、策略和方法。糖蛋白在人胚肾293T细胞中产生，这使得能够快速筛选构建体并以毫克量表达蛋白质。GP与抗体片段（Fab）的复合物促进了结晶，并且尝试了一系列去糖基化策略，包括糖突变体、酶促去糖基化、昆虫细胞表达和聚糖合成途径抑制剂，以改善衍射较弱的糖蛋白晶体。通过确定未复合Fab的结构提高了分子置换搜索模型的信噪比。将Fab模型相位与硒异常信号进行相位组合，随后进行非晶体学对称性（NCS）平均密度修正，得到了清晰可解释的电子密度图。使用B值锐化的电子密度图辅助模型构建，并通过以N - 连接聚糖位点为锚点构建替代序列并结合二级结构预测来确认正确的序列比对。
