Model Building and Refinement of a Natively Glycosylated HIV-1 Env Protein by High-Resolution Cryoelectron Microscopy.
Model Building and Refinement of a Natively Glycosylated HIV-1 Env Protein by High-Resolution Cryoelectron Microscopy.
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DOI：
10.1016/j.str.2015.07.020
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发表时间：
2015-10-06
期刊：
影响因子：
--
通讯作者：
Ward AB
中科院分区：
文献类型：
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作者：
Lee JH;de Val N;Lyumkis D;Ward AB
哺乳动物细胞的分泌和膜蛋白经历翻译后修饰，包括N-连接的糖基化，这可能导致大量可能的糖形式。这种样品的不均一性对于结构研究，特别是X射线结晶学来说可能是个问题。因此，像HIV-1Env病毒刺突蛋白这样的高度糖基化的蛋白质的晶体结构是通过去除大多数糖链来确定的。这一步骤最常使用内切糖苷酶H(EndoH)进行，并要求所有表达的多糖都是高甘露糖形式，而高甘露糖形式通常不是天然的糖形式。随着单颗粒冷冻电子显微镜(CryoEM)技术的显著改进，我们证明现在有可能在4.36？分辨率的溶液中提炼和构建天然糖基化的HIV-1包膜结构。在这个决议下，我们现在可以分析广谱中和抗体(BNab)的完整表位，PGT128，在用天然多糖表达的三聚体的背景下。
Secretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X-ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV-1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high-mannose form, which is often not the native glycoform. With significantly improved technologies in single particle cryo-electron microscopy (cryoEM), we demonstrate that now it is possible to refine and build natively glycosylated HIV-1 Env structures in solution to 4.36 Å resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb), PGT128, in the context of the trimer expressed with native glycans.