2022 Fiscal Year Research-status Report
Architecture and mechanism of the shelterin complex
| Project/Area Number |
21K20645
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
AYALA・HERNANDEZ Rafael 沖縄科学技術大学院大学, 生体分子電子顕微鏡解析ユニット, ポストドクトラルスカラー (00912601)
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| Project Period (FY) |
2021-08-30 – 2024-03-31
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| Keywords | cryo-EM / structural biology / telomeres / cancer / aging / molecular biology |
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| Outline of Annual Research Achievements |
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.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Even though results from initial negative screening looked promising, the transition to cryo-EM for obtaining high-resolution structures has presented unexpected challenges. These can be summarized as a higher heterogeneity in the sample than what was observed in negative staining. Nevertheless, multiple strategies for overcoming the presented difficulties are being implemented, such as the described use of chemical cross-linkers.
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| Strategy for Future Research Activity |
The first strategy to be tested will be the usage of chemical cross-linkers to improve sample stability and homogeneity in cryo-EM grids. Firstly, further attempts at using glutaraldehyde will be carried out, performing before hand a buffer exchange step to remove buffer components incompatible with glutaraldehyde cross-linking. If glutaraldehyde is not succesful, other cross-linkers with different length will be tested, such as bissulfosuccinimidyl suberate.
An alternative strategy will be the addition of a DNA substrate mimicking telomeres and to which shelterin can bind. The binding to such DNA substrate can potentially improve the stability and homogeneity of the complex. Additionally, addition of DNA substrate can be combined with the usage of chemical cross-linkers, or with cross-linking by means of irradiation with ultraviolet light.
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| Causes of Carryover |
As described before, unexpected obstacles in proceeding to high-resolution cryo-EM reconstructions have been found. The incurring amount to be used next fiscal year will be used to cover for the expenses of the detailed strategies to improve the homogeneity and stability of the sample, including the usage of chemical cross-linkers, addition of DNA substrate and usage of UV crosslinking.
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