2015 Fiscal Year Research-status Report
極低温単粒子時分割解析によるFoF1-ATP合成酵素のFo/F1共役機構の研究
| Project/Area Number |
15K14464
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| Research Institution | University of Hyogo |
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Principal Investigator |
Gerle Christoph 兵庫県立大学, 生命理学研究科, 准教授 (10561970)
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| Project Period (FY) |
2015-04-01 – 2017-03-31
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| Keywords | membrane protein / molecular machine / rotational catalysis / mitochondria / cryo-EM / single particle / image processing / structural dynamics |
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| Outline of Annual Research Achievements |
The aim of this project is to use single particle cryo-EM to elucidate the structural dynamics underlying rotational catalysis in mammalian FoF1 ATP synthase. The most basic requirement for this project is the purification of intact, stable, monodisperse and homogenous mitochondrial FoF1 complexes. By employing novel, lipid like detergents we could improve the quality of our sample significantly, which is now at a level sufficient for structure analysis by single particle cryo-EM.
Another critical requirement for this project is the highly reproducible preparation of cryo grids of a quality good enough for high resolution single particle cryo-EM. By applying our newly developed GraDeR method to this project we were able to obtain high resolution compatible cryo grids on a routine basis.
Both improvements allowed us now to start collecting data on a regular basis and receive critical feedback for both purification and specimen preparation.
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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
Though very challenging, both basic requirement of this project (1) purification of suitable FoF1 complexes and (2) the routine preparation of suitable cryo-EM grids for single particle analysis have been fulfilled in the first year. These results allow for optimism that we will be able to improve the structural analysis of bovine FoF1 by single particle cryo-EM to a level, where visualization of the structural dynamics underlying rotational catalysis is feasible.
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| Strategy for Future Research Activity |
Since the sample and specimen requirements for this project have been basically fulfilled, the focus in the second year will be on data acquisition strategy and image processing. The highest possible image quality is required for implementing the sorting of conformational states underlying visualization of structural dynamics by single particle cryo-EM. Therefore we will test various types of direct electron detectors (DED), electron dose exposures and lens settings to arrive at optimal image acquisition settings. Furthermore, small test data sets (<20,000 images) taken under differing settings will be used to test image sorting strategies, before implementing them on large data sets (>1,000,000 images).
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[Journal Article] Two-dimensional crystallization of monomeric bovine cytochrome c oxidase with bound cytochrome c in reconstituted lipid membranes.2016
Author(s)
Osuda, Y., Shinzawa-Itoh, K., Tani, K., Maeda, S., Yoshikawa, S., Tsukihara, T. and Gerle C.
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Journal Title
Microscopy
Volume: 65
Pages: 263-267
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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[Journal Article] Bovine F1Fo ATP synthase monomers bend the lipid bilayer in 2D membrane crystals.2015
Author(s)
Jiko, C., Davies, K.M., Shinzawa-Itoh, K., Tani, K., Maeda, S., Mills, D.J., Tsukihara, T., Fujiyoshi, Y., Kuhlbrandt, W. & Gerle C.
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Journal Title
eLife
Volume: 4
Pages: e06119
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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