Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition
Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition
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DOI：
10.1016/j.jsb.2006.04.014
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发表时间：
2006-11-01
影响因子：
3
通讯作者：
Baumeister, Wolfgang
中科院分区：
文献类型：
--
作者：
Ortiz, Julio O.;Foerster, Friedrich;Baumeister, Wolfgang
冷冻电子断层扫描（CET）将三维（313）成像的潜力与生物样品的近生命保存相结合。它允许检查大型和随机可变结构，如细胞器或整个细胞。在当前的分辨率下，可以在其功能细胞环境中可视化大分子复合物。模式识别方法可用于断层图像的系统解释;基于目标分子的结构特征及其与模板的对应关系来识别和定位目标分子。在这里，我们证明，这样的方法可以用来映射70 S核糖体在一个完整的原核细胞（意大利螺原体）具有高保真度，尽管低信噪比（SNR）的断层图像。在4.7 nm的分辨率下，从断层图像中发现的236个核糖体产生的平均值与通过X射线晶体学或冷冻电子显微镜获得的分离的核糖体的高分辨率结构非常一致。在实验条件下（对数生长期），核糖体均匀分布在整个细胞质中，约占细胞体积的5%。发现约15%的亚组非常接近质膜，并且相对于质膜具有不同的取向。这项研究代表了产生更全面的大分子复合物细胞图谱的第一步。(c)2006年爱思唯尔公司All rights reserved.
Cryoelectron tomography (CET) combines the potential of three-dimensional (313) imaging with a close-to-life preservation of biological samples. It allows the examination of large and stochastically variable structures, such as organelles or whole cells. At the current resolution it becomes possible to visualize large macromolecular complexes in their functional cellular environments. Pattern recognition methods can be used for a systematic interpretation of the tomograms; target molecules are identified and located based on their structural signature and their correspondence with a template. Here, we demonstrate that such an approach can be used to map 70S ribosomes in an intact prokaryotic cell (Spiroplasma melliferum) with high fidelity, in spite of the low signal-to-noise ratio (SNR) of the tomograms. At a resolution of 4.7 nm the average generated from the 236 ribosomes found in a tomogram is in good agreement with high resolution structures of isolated ribosomes as obtained by X-ray crystallography or cryoelectron microscopy. Under the conditions of the experiment (logarithmic growth phase) the ribosomes are evenly distributed throughout the cytosol, occupying approximately 5% of the cellular volume. A subset of about 15% is found in close proximity to and with a distinct orientation with respect to the plasma membrane. This study represents a first step towards generating a more comprehensive cellular atlas of macromolecular complexes. (c) 2006 Elsevier Inc. All rights reserved.