Schaller Research Groups, Department of Infectious Diseases, Virology, Heidelberg University Hospital, Heidelberg, Germany; Research Group "Membrane Biology of Viral Infection", Department of Infectious Diseases, Virology, Heidelberg University Hospital, Heidelberg, Germany.
Schaller Research Groups, Department of Infectious Diseases, Virology, Heidelberg University Hospital, Heidelberg, Germany; Research Group "Membrane Biology of Viral Infection", Department of Infectious Diseases, Virology, Heidelberg University Hospital, Heidelberg, Germany.
J Struct Biol. 2021 Jun;213(2):107742. doi: 10.1016/j.jsb.2021.107742. Epub 2021 May 8.
Cryo-electron tomography (cryo-ET) is a pivotal imaging technique for studying the structure of pleomorphic enveloped viruses and their interactions with the host at native conditions. Owing to the limited tilting range of samples with a slab geometry, electron tomograms suffer from so-called missing wedge information in Fourier space. In dual-axis cryo-ET, two tomograms reconstructed from orthogonally oriented tilt series are combined into a tomogram with improved resolution as the missing wedge information is reduced to a pyramid. Volta phase plate (VPP) allows to perform in-focus cryo-ET with high contrast transfer at low-resolution frequencies and thus its application may improve the quality of dual-axis tomograms. Here, we compare dual-axis cryo-ET with and without VPP on Ebola virus-like particles to visualize and segment viral and host cell proteins within the membrane-enveloped filamentous particles. Dual-axis VPP cryo-ET reduces the missing wedge information and ray artifacts arising from the weighted back-projection during tomogram reconstruction, thereby minimizing ambiguity in the analysis of crowded environments and facilitating 3D segmentation. We show that dual-axis VPP tomograms provide a comprehensive description of macromolecular organizations such as nucleocapsid assembly states, the distribution of glycoproteins on the viral envelope and asymmetric arrangements of the VP40 layer in non-filamentous regions of virus-like particles. Our data reveal actin filaments within virus-like particles in close proximity to the viral VP40 scaffold, suggesting a direct interaction between VP40 and actin filaments. Dual-axis VPP cryo-ET provides more complete 3D information at high contrast and allows for better interpretation of macromolecule interactions and pleomorphic organizations.
冷冻电子断层扫描（cryo-ET）是一种研究多形包膜病毒结构及其在天然条件下与宿主相互作用的关键成像技术。由于具有板状几何形状的样品的倾斜范围有限，电子断层图像在傅立叶空间中会出现所谓的缺失楔形信息。在双轴 cryo-ET 中，两个从正交倾斜系列重建的断层图像组合成一个具有改进分辨率的断层图像，因为缺失楔形信息减少到一个金字塔。伏打相板（VPP）允许在低分辨率频率下进行高对比度转移的聚焦 cryo-ET，因此其应用可以提高双轴断层图像的质量。在这里，我们比较了 Ebola 病毒样颗粒上有和没有 VPP 的双轴 cryo-ET，以可视化和分割膜包裹丝状颗粒内的病毒和宿主细胞蛋白。双轴 VPP cryo-ET 减少了缺失楔形信息和射线伪影，这些伪影是在断层图像重建过程中进行加权反向投影产生的，从而最大限度地减少了在拥挤环境中分析的模糊性，并促进了 3D 分割。我们表明，双轴 VPP 断层图像提供了对核衣壳组装状态等大分子组织的全面描述，以及糖蛋白在病毒包膜上的分布和病毒样颗粒中非丝状区域中 VP40 层的不对称排列。我们的数据显示，病毒样颗粒内的肌动蛋白纤维与病毒 VP40 支架非常接近，表明 VP40 和肌动蛋白纤维之间存在直接相互作用。双轴 VPP cryo-ET 以高对比度提供更完整的 3D 信息，并允许更好地解释大分子相互作用和多形组织。
