Architecture and mechanism of the shelterin complex

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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.