Oral Presentation 2014 International Biophysics Congress

Structural analysis of the 26S proteasome by cryo-electron microscopy and single-particle analysis (#58)

Zhuo Wang 1 , Yasuo Okuma 1 , Daiske Kasuya 2 , Kaoru Mitsuoka 3 , Yasushi Saeki 3 , Takuo Yasunaga 1
  1. Department of Bioscience and Bioinformatics, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, Iizuka, Japan
  2. Biomedicinal Information Research Center, Japan Biological Information Consortium (JBIC), Tokyo, Japan
  3. Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan

In eukaryotic cells, the ubiquitin proteasome system (UPS) is responsible for selective protein degradation. In this system, the 26S proteasome plays a very important role to degrade target proteins which are ubiquitinated by ubiquitin ligases. The 2.5-MDa molecular machine comprises two types of subcomplexes: the barrel 20S core particle (CP) and the two 19S regulatory particles (RP), which associate with the CP and prepare substracts for degradation. Since the weak assembly of the 26S makes structural analysis more difficult, we added 10% glycerol in the specimen for stabilization. But the low-contrast which made ‘pick up’ more difficult has been found in the 26S photograms. Thus we used the Gradient Fixation (GraFix) method to purify and stabilize the 26S. After using GraFix, Not only the glycerol which made the low-contrast could be easy removed, but also the mount of 26S proteasome has been increased in the specimen. Despite removed the glycerol, the EM images which have been taken under the influence of cryogenic temperature environment are still lacking in visibility. In order to solve this problem, CTF compensation using Wiener filter which can remove the additive noise and invert the blurring simultaneously, has been used in this research. As a result, 26S single particles could be visible and easy to be picked up. The refined 3D maps and comparison between wildtype and Rpn10 mutant will be reported here.