Architecture and mechanism of the shelterin complex

用低温电子显微镜分析人类避难所复合体的尝试已经完成。已经找到了适当地将络合物沉积到低温EM网格上的条件，并收集了多个低温EM数据集。然而，经过详细的分析，已经发现这些颗粒不够均匀，不足以获得高分辨率结构。为了改善样品的均一性，执行了更严格的纯化方案，包括最后的凝胶过滤步骤，并对来自单个凝胶过滤峰(经SDS-PAGE证实包含络合物的所有组分)的络合物进行成像。凝胶过滤洗脱后立即准备网格，以排除样品冻结和解冻循环造成的负面影响。然而，这并没有导致样本的改善。观察到的异质性的另一个潜在原因是冷冻-EM网格冻结时复合体的解离或分解。为了克服这个问题，一组化学交联剂正在进行测试。这种化学交联剂在络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。已经用戊二醛进行了初步测试。然而，由于存在与戊二醛交联(Tris)不相容的缓冲成分，结果不确定。

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.