研究課題
In the spirit of the Hoga Kakenhi grant this project aims high at using single particle cryo-EM to obtain a time resolved structural analysis of the mammalian F-ATP synthase during rotary catalysis. Structural dynamics of rotary catalysis is an an important scientific problem. It is difficult to tackle by X-ray crystallography and ways and means to solve this problem will be influential for improving the understanding of other membrane bound molecular machines. We have succeeded in establishing a mild purification procedure and importantly innovated a new sample preparation method for the use of cryo-EM of fragile membrane complexes termed GraDeR. We successfully applied GraDeR to the cryo-EM analysis of mammalian F-ATP synthase and obtained new insights into the structure of this important mitochondrial energy converter. Furthermore, we were able to apply focused masking to cryoEM image data sets to resolve critical dynamics in the transmembrane Fo domain. Taken together, the foundations of using time resolved cryoEM for analyzing the structural dynamics of mammalian F-ATP synthase were firmly established and thus the door for a whole new series of cryo-EM studies on mammalian F-ATP synthase opened.
The pdf of the step-by-step protocol has been already downloaded several hundred times and the GraDeR web-page is well known in the community.
すべて 2016 その他
すべて 雑誌論文 (3件) (うち国際共著 2件、 査読あり 3件、 オープンアクセス 2件、 謝辞記載あり 1件) 学会発表 (6件) (うち国際学会 3件、 招待講演 4件) 備考 (1件)
Microscopy
巻: 65 ページ: 263-267
doi: 10.1093/jmicro/dfv381
BBA-Bioenergetics
巻: 1857 ページ: 1191-1196
http://dx.doi.org/10.1016/j.bbabio.2016.03.008
生化学
巻: 88 ページ: 532-536
10.14952/SEIKAGAKU.2016.880532
https://grader-protocol.com/
