Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC, USA.
Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA.
Nat Commun. 2021 Mar 30;12(1):1957. doi: 10.1038/s41467-021-22251-8.
Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.
对在电子显微镜中成像的冷冻保存标本进行断层重建，然后提取和平均子体积，已成功用于在其生物环境中获得大分子的原子模型。与其他技术所需的生化分离步骤相比，该方法使细胞能够进行原位结构研究。然而，需要补偿在机械导航标本过程中引入的靶向误差，这极大地降低了断层重建数据采集的速度，从而限制了其实用价值。在这里，我们引入了倾斜系列采集和处理的方案，通过使用束流图像移位将每个阶段位置成像的区域数量增加一个数量级，通过在成像过程中集成几何约束来实现高精度的靶向，并通过执行每个倾斜度的像散 CTF 估计和数据驱动的曝光加权来提高最终的地图分辨率。我们通过确定低分子量靶标（~300 kDa）的结构来验证我们的束流图像移位电子低温断层扫描（BISECT）方法，在 3.6 Å 的分辨率下可以清楚地分辨出单个侧链的密度。
