Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography.
Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography.
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DOI：
10.1038/s41467-021-22251-8
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发表时间：
2021-03-30
影响因子：
16.6
通讯作者：
Bartesaghi A
中科院分区：
文献类型：
--
作者：
Bouvette J;Liu HF;Du X;Zhou Y;Sikkema AP;da Fonseca Rezende E Mello J;Klemm BP;Huang R;Schaaper RM;Borgnia MJ;Bartesaghi A
在电子显微镜下成像的冷冻标本的断层扫描重建，然后提取和平均子体积，已成功用于推导生物环境中大分子的原子模型。该方法消除了其他技术所需的生化分离步骤，使细胞能够进行原位结构研究。然而，需要补偿样本机械导航期间引入的瞄准误差，这显着减慢了断层扫描数据收集的速度，从而限制了其实际价值。在这里，我们介绍了倾斜系列采集和处理协议，与现有方法相比，该协议可将数据收集速度加快一个数量级，并提高地图分辨率。我们通过使用光束图像移位来倍增每个平台位置成像的区域数量，通过在成像过程中集成几何约束以实现高精度瞄准，并通过执行每倾斜像散 CTF 估计和数据驱动的曝光加权来提高最终图分辨率来实现这一目标。我们通过在 3.6 Å 分辨率下确定低分子量靶标 (~300 kDa) 的结构来验证我们的光束图像移位电子冷冻断层扫描 (BISECT) 方法，其中各个侧链的密度清晰可见。冷冻保存标本的断层扫描重建可以进行原位结构研究。在这里，作者提出了束像位移电子冷冻断层扫描 (BISECT) 方法，与早期方法相比，该方法可加快数据收集速度并提高图谱分辨率，并以 3.6 Å 分辨率解析了 300 kDa 蛋白质复合物的体外结构作为测试案例。
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. Tomographic reconstructions of cryopreserved specimens enable in-situ structural studies. Here, the authors present the beam image-shift electron cryo-tomography (BISECT) approach that accelerates data collection speed and improves the map resolution compared to earlier approaches and present the in vitro structure of a 300 kDa protein complex that was solved at 3.6 Å resolution as a test case.
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影响因子：
2.2
作者：
Fernandez, J. J.;Li, S.;Crowther, R. A.
通讯作者：
Crowther, R. A.
影响因子：
3
作者：
Chreifi, Georges;Chen, Songye;Jensen, Grant J.
通讯作者：
Jensen, Grant J.
影响因子：
3
作者：
Bartesaghi, A.;Sprechmann, P.;Subramaniam, S.
通讯作者：
Subramaniam, S.
影响因子：
3
作者：
Mastronarde, DN
通讯作者：
Mastronarde, DN
影响因子：
3
作者：
Su, Min
通讯作者：
Su, Min