2019 Fiscal Year Research-status Report
Towards a new type of antibiotic: elucidation of the activation signal and signal transduction mechanism of the type III secretion system of Shigella flexneri
| Project/Area Number |
19K16661
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
CHEUNG Martin 沖縄科学技術大学院大学, 量子波光学顕微鏡ユニット, 研究員 (90832163)
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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Keywords | Shigelle flexneri / T3SS / Tip complex / Cryo-EM |
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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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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| Strategy for Future Research Activity |
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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| Causes of Carryover |
Work conducted hitherto represents the first phase of the project. During the next fiscal year, we will continue to the second phase of the project which involves using the methods and techniques developed in the first phase to achieve a 5 Angstrom 3D reconstruction of the T3SS tip complex.
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