Microscopy, 2014, Vol. 63, No. S1
i32
Structural analysis of the 26S proteasome by cryo-electron microscopy and Single-Particle Analysis Zhuo Wang1, Yasuo Okuma1, Daiske Kasuya2, Kaoru Mitsuoka3, Yasushi Saeki4, and Takuo Yasunaga1,5 1
Department of Bioscience and Bioinformatics, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, 2 Biomedicinal Information Research Center, Japan Biological Information Consortium (JBIC), 3Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, 4Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, and 5JST, SENTAN
References 1. Nishino Y., Yasunaga T., Miyazawa A (2007) A genetically encoded metallothionein tag enabling efficient protein detection by electron microscopy, Journal of Electron Microscopy, 56(3), 93–101 2. Park T.,J., Lee S.,Y., Heo N.,S., Seo T.S (2010) In Vivo Synthesis of Diverse Metal Nanoparticles by Recombinant Escherichia coli, Angewandte Chemie International Edition, 49, 7019–7024 3. Haru-aki Yanagisawa Garrison, Mathis Toshiyuki, Oda Masafumi, Hirono Elizabeth A., Richey Hiroaki, Ishikawa Wallace F., Marshall Masahide, Hongmin Qin Kikkawa (2014) FAP20 is an inner junction protein of doublet microtubules essential for both the planar asymmetrical waveform and stability of flagella in Chlamydomonas, Mol. Biol. Cell May 1, 2014 vol. 25 no. 9 1472–1483 doi: 10.1093/jmicro/dfu065
Development of a user-friendly system for image processing of electron microscopy by integrating a web browser and PIONE with Eos
doi: 10.1093/jmicro/dfu062 Takafumi Tsukamoto1 and Takuo Yasunaga1,2 1
Metallothionein labeling for CLEM method for identification of protein subunits Ryutaro Yamanaka1, Yuka Hirasaka1, Mingyue Jin1, Haruaki Yanagisawa2, and Takuo Yasunaga1,3 1 Kyushu Institute of Technology, 2Univ. of Tokyo, and 3JST, SENTAN CLEM (correlative light and electron microscopy) is one of the powerful techniques to elucidate the localization and structure of the
Dept. of Bioscience and Bioinformatics, Faculty of Computer Science and Systems Enginering, Kyushu Institute of Technology, and 2 SENTAN, JST 680-4 Kawazu, Iizuka, Fukuoka, Japan Eos (Extensible object-oriented system) is one of the powerful applications for image processing of electron micrographs. In usual cases, Eos works with only character user interfaces (CUI) under the operating systems (OS) such as OS-X or Linux, not user-friendly. Thus, users of Eos need to be expert at image processing of electron micrographs, and have a little knowledge of computer science, as well. However, all the persons who require Eos does not an expert for CUI.
Downloaded from http://jmicro.oxfordjournals.org/ at University of Manitoba on April 24, 2015
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 is composed of a barrel shaped 20S core particle (20S-CP) harbouring the proteolytic chamber together with one or two 19S regulatory particles (19S-RP), which associate with the 20S-CP. The 20S-CP consists of heptameric rings, which are named α and β. The four stacked rings of seven highly homologous subunits arranged as a α7-β7-β7-α7 barrel. The 20S-CP is like a factory which destroys ubiquitinated substrates. Both ends of the 20S-CP connect with the 19S-RP, which is composed of at least 18 subunits and is responsible for the recognition, deubiquitination, engagement, unfolding and translocation of ubiquitinated substrates. 19S-RP has been divided into two biochemically subcomplexes-base and lid. The base consists of a ring of six AAA-ATPase subunits, Rpt1-Rpt2-Rpt6-Rpt3-Rpt4-Rpt5, together with the non-ATPase subunits Rpn1, Rpn2 and uibiqutin receptor, Rpn13. The Rpt assembly constitutes an ATP-dependent motor to unfold and translate the substrate proteins into 20S-RP. Rpn1 and Rpn2, two scaffolds which share significant sequence homology, are the largest subunits in 19S-RP. The lid has two group, PCI (Rpn3, Rpn5-7, and Rpn12) and MPN (Rpn8 and Rpn11). Here, Rpn11 is regarded as the deubiquitinating enzyme. However, the position of the second ubiquitin receptor, Rpn10, is still unclear. In our study, we used the GraFix (Gradient Fixation) method to purify and stabilize holoenzyme 26S proteasome for structural analysis. After using GraFix, not only could the glycerol which was contributed to the low contrast of EM images be easy removed, but also the mount of 26S proteasome could be increased in the specimen. However, despite the glycerol remove, the EM images in the cryogenic temperature environment are still lacking in visibility. Even after CTF (Contrast Transfer Function) correction, the images are still hard to pick-up. In order to solve this problem, the Wiener filter which can remove the additive noise and invert the blurring simultaneously, was used in this research. According the defocus value, the frequency characteristic of the image information signal can be changed. Low defocus value makes the frequency-domain higher. This means that the detail of the image information can be visible. In other words, high defocus value creates a low frequency-domain, so the contrast of the whole image can be high. So here, we used a pair of images with different defocus value (high and low) in the same position to fix into Winner filter. As a result, we got higher contrast images which were easy to pick up. And the single particles of wildtype and the Rpn10 mutant of 26S proteasome (totally 2111 and 544) were used to obtain 3D reconstruction structures. Finally, we also compared both 3D structures to define the Rpn10 position.
target proteins or their complexes in cell. First, target proteins labeled fluorescently can be searched using a fluorescence microscope, i.e., due to its low resolution (200nm), it is used as rough searching of target proteins. After rough detection of the localization of target proteins, they can be easily observed by electron microscopy with a high resolution and processed into fine structure, especially 3D structure. On the other hand, in the case of only electron microscopy, it is difficult for researchers to detect their localization due to a narrow range of views and no labeling of them. Thus, CLEM normally needs fluorescent labels for fluorescence microscopy but a label for electron microscopy is also expectedly for easier detection. Thus we focused on metallothionein. Metallothionein binds to cadmium ions, i.e., heavy atoms with strong density in electron microscopy [1]; in addition, cadmium ions and selenium ions are known to form Qdot-like nanoparticles induced by metallothionein [2]. These are 2 ∼ 5nm in size, fluorescent wavelength changes depending on the size of nanoparticles. Thus, target proteins fused with metallothionein could be observed by both of fluorescence microscopy and electron microscopy. We here used Chlamydomonas reinhardtii, single cell green algae with two flagella. Flagella are used for bending motion and motility. Flagella contain FAP20 (Flagellar Asociate Protein 20) and PACRG (PArkin Co-Regulated gene), which are related to composing axoneme architecture. If Chlamydomonas reinhardtii doesn’t have FAP20 or PACRG, they can’t generate bending motion. It is considered that FAP20 and PACRG locate on the root of the radial spoke. Recently the location of FAP20 was reported by Yanagisawa et al.[3]. First, we also focus on detecting localization of FAP20 and then will do so on that of PACKRG. We could observe fluorescence of metallothionein fused with FAP20 to form nanoparticle. We are now trying to observe larger electron density from metallothionein with cadmium for CLEM.
i32
Structural analysis of the 26S proteasome by cryo-electron microscopy and Single-Particle Analysis Zhuo Wang1, Yasuo Okuma1, Daiske Kasuya2, Kaoru Mitsuoka3, Yasushi Saeki4, and Takuo Yasunaga1,5 1
Department of Bioscience and Bioinformatics, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, 2 Biomedicinal Information Research Center, Japan Biological Information Consortium (JBIC), 3Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, 4Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, and 5JST, SENTAN
References 1. Nishino Y., Yasunaga T., Miyazawa A (2007) A genetically encoded metallothionein tag enabling efficient protein detection by electron microscopy, Journal of Electron Microscopy, 56(3), 93–101 2. Park T.,J., Lee S.,Y., Heo N.,S., Seo T.S (2010) In Vivo Synthesis of Diverse Metal Nanoparticles by Recombinant Escherichia coli, Angewandte Chemie International Edition, 49, 7019–7024 3. Haru-aki Yanagisawa Garrison, Mathis Toshiyuki, Oda Masafumi, Hirono Elizabeth A., Richey Hiroaki, Ishikawa Wallace F., Marshall Masahide, Hongmin Qin Kikkawa (2014) FAP20 is an inner junction protein of doublet microtubules essential for both the planar asymmetrical waveform and stability of flagella in Chlamydomonas, Mol. Biol. Cell May 1, 2014 vol. 25 no. 9 1472–1483 doi: 10.1093/jmicro/dfu065
Development of a user-friendly system for image processing of electron microscopy by integrating a web browser and PIONE with Eos
doi: 10.1093/jmicro/dfu062 Takafumi Tsukamoto1 and Takuo Yasunaga1,2 1
Metallothionein labeling for CLEM method for identification of protein subunits Ryutaro Yamanaka1, Yuka Hirasaka1, Mingyue Jin1, Haruaki Yanagisawa2, and Takuo Yasunaga1,3 1 Kyushu Institute of Technology, 2Univ. of Tokyo, and 3JST, SENTAN CLEM (correlative light and electron microscopy) is one of the powerful techniques to elucidate the localization and structure of the
Dept. of Bioscience and Bioinformatics, Faculty of Computer Science and Systems Enginering, Kyushu Institute of Technology, and 2 SENTAN, JST 680-4 Kawazu, Iizuka, Fukuoka, Japan Eos (Extensible object-oriented system) is one of the powerful applications for image processing of electron micrographs. In usual cases, Eos works with only character user interfaces (CUI) under the operating systems (OS) such as OS-X or Linux, not user-friendly. Thus, users of Eos need to be expert at image processing of electron micrographs, and have a little knowledge of computer science, as well. However, all the persons who require Eos does not an expert for CUI.
Downloaded from http://jmicro.oxfordjournals.org/ at University of Manitoba on April 24, 2015
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 is composed of a barrel shaped 20S core particle (20S-CP) harbouring the proteolytic chamber together with one or two 19S regulatory particles (19S-RP), which associate with the 20S-CP. The 20S-CP consists of heptameric rings, which are named α and β. The four stacked rings of seven highly homologous subunits arranged as a α7-β7-β7-α7 barrel. The 20S-CP is like a factory which destroys ubiquitinated substrates. Both ends of the 20S-CP connect with the 19S-RP, which is composed of at least 18 subunits and is responsible for the recognition, deubiquitination, engagement, unfolding and translocation of ubiquitinated substrates. 19S-RP has been divided into two biochemically subcomplexes-base and lid. The base consists of a ring of six AAA-ATPase subunits, Rpt1-Rpt2-Rpt6-Rpt3-Rpt4-Rpt5, together with the non-ATPase subunits Rpn1, Rpn2 and uibiqutin receptor, Rpn13. The Rpt assembly constitutes an ATP-dependent motor to unfold and translate the substrate proteins into 20S-RP. Rpn1 and Rpn2, two scaffolds which share significant sequence homology, are the largest subunits in 19S-RP. The lid has two group, PCI (Rpn3, Rpn5-7, and Rpn12) and MPN (Rpn8 and Rpn11). Here, Rpn11 is regarded as the deubiquitinating enzyme. However, the position of the second ubiquitin receptor, Rpn10, is still unclear. In our study, we used the GraFix (Gradient Fixation) method to purify and stabilize holoenzyme 26S proteasome for structural analysis. After using GraFix, not only could the glycerol which was contributed to the low contrast of EM images be easy removed, but also the mount of 26S proteasome could be increased in the specimen. However, despite the glycerol remove, the EM images in the cryogenic temperature environment are still lacking in visibility. Even after CTF (Contrast Transfer Function) correction, the images are still hard to pick-up. In order to solve this problem, the Wiener filter which can remove the additive noise and invert the blurring simultaneously, was used in this research. According the defocus value, the frequency characteristic of the image information signal can be changed. Low defocus value makes the frequency-domain higher. This means that the detail of the image information can be visible. In other words, high defocus value creates a low frequency-domain, so the contrast of the whole image can be high. So here, we used a pair of images with different defocus value (high and low) in the same position to fix into Winner filter. As a result, we got higher contrast images which were easy to pick up. And the single particles of wildtype and the Rpn10 mutant of 26S proteasome (totally 2111 and 544) were used to obtain 3D reconstruction structures. Finally, we also compared both 3D structures to define the Rpn10 position.
target proteins or their complexes in cell. First, target proteins labeled fluorescently can be searched using a fluorescence microscope, i.e., due to its low resolution (200nm), it is used as rough searching of target proteins. After rough detection of the localization of target proteins, they can be easily observed by electron microscopy with a high resolution and processed into fine structure, especially 3D structure. On the other hand, in the case of only electron microscopy, it is difficult for researchers to detect their localization due to a narrow range of views and no labeling of them. Thus, CLEM normally needs fluorescent labels for fluorescence microscopy but a label for electron microscopy is also expectedly for easier detection. Thus we focused on metallothionein. Metallothionein binds to cadmium ions, i.e., heavy atoms with strong density in electron microscopy [1]; in addition, cadmium ions and selenium ions are known to form Qdot-like nanoparticles induced by metallothionein [2]. These are 2 ∼ 5nm in size, fluorescent wavelength changes depending on the size of nanoparticles. Thus, target proteins fused with metallothionein could be observed by both of fluorescence microscopy and electron microscopy. We here used Chlamydomonas reinhardtii, single cell green algae with two flagella. Flagella are used for bending motion and motility. Flagella contain FAP20 (Flagellar Asociate Protein 20) and PACRG (PArkin Co-Regulated gene), which are related to composing axoneme architecture. If Chlamydomonas reinhardtii doesn’t have FAP20 or PACRG, they can’t generate bending motion. It is considered that FAP20 and PACRG locate on the root of the radial spoke. Recently the location of FAP20 was reported by Yanagisawa et al.[3]. First, we also focus on detecting localization of FAP20 and then will do so on that of PACKRG. We could observe fluorescence of metallothionein fused with FAP20 to form nanoparticle. We are now trying to observe larger electron density from metallothionein with cadmium for CLEM.
