MRC-University of Glasgow Centre for Virus Research, Sir Michael Stoker Building, Garscube Campus, 464 Bearsden Road, Glasgow, G61 1QH, Scotland, UK.
Sci Rep. 2020 Oct 19;10(1):17596. doi: 10.1038/s41598-020-74104-x.
Cryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures approaching atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (> 500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised and costly equipment. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions confirm that the capsid associated tegument complex is present on capsids prior to nuclear egress. We demonstrate that this method is suited to both 3D structure determination and correlative light/electron microscopy, thus expanding the scope of cryogenic cellular imaging.
低温电子显微镜（cryo-EM）是一种用于结构测定的关键方法，涉及对嵌入玻璃态冰中的纯化物质进行成像。然后对图像进行计算处理，以获得接近原子分辨率的三维结构。人们越来越感兴趣的是通过 cryo-EM 将结构研究扩展到细胞中，在细胞中可以在背景下对生物结构和过程进行成像。然而，电子的有限穿透能力阻止了对厚标本（>500nm）的成像。为克服这一问题而采用的 cryo 切片方法在技术上具有挑战性，容易产生伪影或涉及专门且昂贵的设备。在这里，我们描述了第一个通过来自真核细胞核的子断层平均法从疱疹病毒衣壳中确定的结构，这是通过 Tokuyasu 方法制备的再玻璃化细胞切片的 cryo 电子断层扫描（cryo-ET）实现的。我们的重建证实，在核外溢之前，衣壳相关的被膜复合物就存在于衣壳上。我们证明该方法既适合 3D 结构测定，也适合相关的光/电子显微镜，从而扩展了低温细胞成像的范围。
