Department of Biological Sciences and Centre for BioImaging Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543.
Gigascience. 2019 Jun 1;8(6). doi: 10.1093/gigascience/giz077.
Cells are powered by a large set of macromolecular complexes, which work together in a crowded environment. The in situ mechanisms of these complexes are unclear because their 3D distribution, organization, and interactions are largely unknown. Electron cryotomography (cryo-ET) can address these knowledge gaps because it produces cryotomograms-3D images that reveal biological structure at ∼4-nm resolution. Cryo-ET uses no fixation, dehydration, staining, or plastic embedment, so cellular features are visualized in a life-like, frozen-hydrated state. To study chromatin and mitotic machinery in situ, we subjected yeast cells to genetic and chemical perturbations, cryosectioned them, and then imaged the cells by cryo-ET.
Here we share >1,000 cryo-ET raw datasets of cryosectioned budding yeast Saccharomyces cerevisiaecollected as part of previously published studies. These data will be valuable to cell biologists who are interested in the nanoscale organization of yeasts and of eukaryotic cells in general. All the unpublished tilt series and a subset of corresponding cryotomograms have been deposited in the EMPIAR resource for the community to use freely. To improve tilt series discoverability, we have uploaded metadata and preliminary notes to publicly accessible Google Sheets, EMPIAR, and GigaDB.
Cellular cryo-ET data can be mined to obtain new cell-biological, structural, and 3D statistical insights in situ. These data contain structures not visible in traditional electron-microscopy data. Template matching and subtomogram averaging of known macromolecular complexes can reveal their 3D distributions and low-resolution structures. Furthermore, these data can serve as testbeds for high-throughput image-analysis pipelines, as training sets for feature-recognition software, for feasibility analysis when planning new structural-cell-biology projects, and as practice data for students.
细胞由大量大分子复合物提供动力，这些复合物在拥挤的环境中协同工作。这些复合物的原位机制尚不清楚，因为其三维分布、组织和相互作用在很大程度上是未知的。电子低温断层扫描（cryo-ET）可以解决这些知识空白，因为它可以生成 cryotomograms-揭示生物学结构的 3D 图像，分辨率约为 4nm。cryo-ET 不使用固定、脱水、染色或塑料包埋，因此细胞特征以类似于生活的、冷冻水合状态可视化。为了在原位研究染色质和有丝分裂机制，我们对酵母细胞进行了遗传和化学扰动，然后对其进行 cryosection 并通过 cryo-ET 对细胞进行成像。
在这里，我们分享了超过 1000 个来自已发表研究的部分 cryosectioned 出芽酵母酿酒酵母 Saccharomyces cerevisiae 的 cryo-ET 原始数据集。这些数据将对细胞生物学家非常有价值，他们对酵母和一般真核细胞的纳米级组织感兴趣。所有未发表的倾斜系列和相应 cryotomograms 的子集已被存入 EMPIAR 资源库，供社区免费使用。为了提高倾斜系列的可发现性，我们已经将元数据和初步注释上传到公共可访问的 Google Sheets、EMPIAR 和 GigaDB。
细胞 cryo-ET 数据可以被挖掘以获得新的细胞生物学、结构和原位 3D 统计洞察力。这些数据包含在传统电子显微镜数据中不可见的结构。已知大分子复合物的模板匹配和亚结构平均化可以揭示它们的 3D 分布和低分辨率结构。此外，这些数据可以作为高通量图像分析管道的测试平台，作为特征识别软件的训练集，用于规划新的结构细胞生物学项目的可行性分析，以及作为学生的实践数据。
