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Architecture and mechanism of the shelterin complex

庇护所综合体的结构和机制

基本信息

批准号：
21K20645
负责人：
AYALA・HERNANDEZ Rafael
金额：
$ 2万
依托单位：
Okinawa Institute of Science and Technology Graduate University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Research Activity Start-up
财政年份：
2021
资助国家：
日本
起止时间：
2021-08-30 至 2024-03-31
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-21K20645/
关键词：
cryo-EM structural biology telomeres cancer aging molecular biology cryoEM chromatin Chromatin Cryo-EM Telomeres DNA damage

项目摘要

Attempts at analyzing human shelterin complex by cryo-EM have been performed. Conditions under which the complex is properly deposited onto cryo-EM grids have been found, and multiple cryo-EM datasets have been collected. However, upon detailed analysis, the particles have been found to not be sufficiently homogeneous for obtaining a high-resolution structure. In order to try to improve the homogeneity of the sample, a stricter purification protocol comprising a final gel filtration step was performed, and complexes from an individual gel filtration peak (confirmed by SDS-PAGE to contain all components of the complex) were imaged. Grids were prepared immediately after elution of gel filtration, to rule out negative effects due to freezing and thawing cycles of the sample. However, this did not lead to an improved sample.Another potential reason for the observed heterogeneity is dissociation or break-up of the complex upon freezing of cryo-EM grids. In order to overcome this problem, a set of chemical cross-linkers is being tested. Such chemical cross-linkers introduce additional covalent bonds in the complex, making it more stable and able to stand the grid freezing process. Initial tests with glutaraldehyde have been performed. However, due to the presence of buffer components incompatible with glutaraldehyde cross-linking (Tris), results were inconclusive.

已尝试通过冷冻电镜分析人shelterin复合物。已经发现复合物适当地沉积到低温EM网格上的条件，并且已经收集了多个低温EM数据集。然而，在详细分析后，发现颗粒对于获得高分辨率结构而言不够均匀。为了尝试改善样品的均匀性，进行了包括最终凝胶过滤步骤的更严格的纯化方案，并对来自单个凝胶过滤峰的复合物（通过SDS-PAGE确认含有复合物的所有组分）进行成像。在凝胶过滤洗脱后立即制备网格，以排除由于样品冻融循环引起的负面影响。观察到异质性的另一个潜在原因是冷冻EM网格时复合物的解离或破裂。为了克服这个问题，正在测试一套化学交联剂。这种化学交联剂在复合物中引入了额外的共价键，使其更稳定，能够经受住网格冻结过程。已使用戊二醛进行初步试验。然而，由于存在与戊二醛交联（Tris）不相容的缓冲液组分，结果不确定。