| 研究実績の概要 |
Recent technical advances in single particle cryo-EM data acquisition, classification, and reconstruction have caused the “EM resolution revolution” within the last few years. Thus, we have been collaborating with Dr. Qiu-xing Jiang at UT Southwestern to reconstruct the three dimensional structures of the Drosophila RLC complex using cryo-EM. To date, we have obtained low resolution EM structure of Dicer-2-R2D2, Dicer-2-R2D2 complex with siRNA, and Dicer-2-R2D2 and TAF11 in complex with siRNA (RLC). Comparison of the EM maps reveals that Dicer-2-R2D2 complex exists in monomeric and dimeric states. The conformation of the RLC is clearly different from that of Dicer-2-R2D2-siRNA complex, suggesting that TAF11 binding results in significant conformational changes of the RLC, which may be critical for the RISC loading activity. However, we encountered the difficulty of protein aggregation and conformation heterogeneity during the cryo-EM reconstruction. We are making good progress to optimize the condition to achieve high-resolution EM reconstructions of Dicer-2-R2D2-siRNA and Dicer-2-R2D2-TAF11-siRNA complexes by obtaining more homogeneous samples, increasing the size of EM dataset, and reducing conformation heterogeneity by sorting EM images (unpublished).
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