| 研究実績の概要 |
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.
|
| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
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.
|
| 今後の研究の推進方策 |
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).
|
