Crystal structure analysis of multi-subunit IFT-B core complex involved in ciliogenesis

• Project/Area Number: 18K06099
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 43020:Structural biochemistry-related
• Research Institution: High Energy Accelerator Research Organization
• Principal Investigator: Kawasaki Masato 大学共同利用機関法人高エネルギー加速器研究機構, 物質構造科学研究所, 准教授 (00342600)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2020: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2018: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 繊毛内輸送 / 多サブユニット複合体 / 結晶構造解析

Outline of Final Research Achievements

The Intraflagellar transport (IFT)-B complex is involved in anterograde ciliary trafficking along the axoneme. Among 16 subunits of the IFT-B complex, IFT74-81 and IFT22-74-81 subcomplexes have been successfully expressed in E. coli using SUMO-tag and coexpression system. However, crystallization trials of the purified subcomplexes failed because of rapid protein degradation within a few days. By quick sample freezing after purification, a rod-like structure of IFT74-81 subcomplex was observed by cryo-EM single particle analysis.

Academic Significance and Societal Importance of the Research Achievements

本研究のSUMOタグを利用した共発現系によって、発現の困難だった多サブユニット複合体の発現が可能になった。この手法はX線結晶構造解析だけでなくクライオ電子顕微鏡単粒子解析にも有効である。