| 研究実績の概要 |
Successfully completed the first phase of the project, which involved image acquisition and three-dimensional (3D) reconstruction of the wild type Shigella flexneri type III secretion system (T3SS) tip complex. T3SSs in near-native states were purified and imaged in the Talos Arctica cryo-TEM, with approximately 17,500 micrographs collected. From these, approximately 70,000 images of T3SSs (i.e. particles) of adequate quality were extracted.
The extracted particles formed the initial data stack. Processing of the data stack, which included particle picking, CTF correction, and motion correction, was initially conducted in Relion 2.1. Due to inherent needle length variability, the precise position of the tip complex within each image varied significantly. In order to correct for this, a bespoke algorithm was performed in program Spider, successfully correcting for this discrepancy. In order to generate an initial ab initio 3D electron density map, a variety of programs were tested. Relion 2.1 was found to perform poorly in this task, as did cisTEM.
Ultimately, program cryoSPARC gave the best results, producing the best 2D classification results and initial 3D map. CryoSPARC was subsequently used for map refinement, generating a final electron density map of just below 10 Angstrom resolution, thus achieving the target of a ‘sub-nanometer’ resolution reconstruction of the tip complex.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
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理由
All stages of the project progressed as planned. The goal before commencing the project was to achieve a 3D reconstruction resolution of below 1 nanometer, which as been accomplished. Ideally, a resolution nearer to 5 Angstrom would be the final target but for the first phase of the project, the results are as expected.
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| 今後の研究の推進方策 |
With having successfully completed all stages of the project, it will be possible to repeat the process but with a view of achieving a higher resolution. All steps are easily adjustable to achieve this goal. In the next phase of the project, the aim will be to achieve a 3D reconstruction resolution of approximately 5 Angstrom.
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