平成 25 年 3 月
放射光システム生物学研究グループ 最終レビュー報告
Synchrotron Radiation System Biology Research Group Final Review

日時 Date：平成 25 年 2 月 メールレビュー February 2013 by e-mailレビュアー Reviewers：
Dr. Midori Kamimura / 上村 みどり(Pharmaceutical Discovery Research Laboratories, Teijin Pharma Limited)
Dr. Hiroyuki Osada /長田 裕之(Chemical Biology Department, RIKEN Advanced Science Institute (ASI))
Prof. Masaru Tanokura / 田之倉 優(Laboratory of Structural Biology and Food Biotechnology, Department of Applied Biological Chemistry,
Graduate School of Agricultural and Life Sciences, The University of Tokyo)
Prof. Tomitake Tsukihara / 月原 冨武(University of Hyogo / Institute for Protein Research, Osaka University)
Dr. So Iwata /岩田 想(SACLA Science Research Group, RIKEN SPring-8 Center (RSC))

評価結果の概要 General comments：[REVIEWER 1]
Goal setting: Dr. Kuramitsu is a leading pioneer in science launched the project of “Whole-cell system of Thermus
thermophiles” to understand all the 3D structures of proteins and reveal the functions and life phenomena of theproteins by biophysical methods using an extremely thermophilic bacterium, Thermus thermophilus HB8 as amodel organism. This research is a one of significant pillars as utilizing SPring-8 for research. Also the project
would not achieved the results without SPring-8 and the significance of SPring-8 became widely known bycarrying it out, therefore his accurate goal setting is highly appreciated.

Research achievements: During the targeted period of this project outcome evaluation, this group, consisting of
three teams such as Functomics Integration Research Team, Functomics Biology I Research Team, andFunctomics Biology II Research Team (Kumamitsu, Shinkai, and Bessho, team leaders respectively) carriedthrough the research. As the group as a whole, despite the smaller numbers of research staff mainly affiliated withRIKEN, more than 20 papers have been published annually. Especially as recent research outcomes, the DNA
Modification Enzymes for structural biological research press release on March 2010 and November 2011 reallyattracted/grabbed attention.
Mainly, on the project of “Whole-cell system of Thermus thermophiles”, as the purpose of analyzing all thefunctions of proteins by genes, Team Kuramitsu constructed and developed new platforms which reach thepurification, crystallization, and analysis of the proteins. Especially development of protein expression plasmids
and their analysis data are compiled as database in order to disclose to the public, so that it seems helping a wide
range of researchers.
Teams of Shinkai and Bessho are proceeding to target molecules related to transcription, translation, andmodification which are important for organisms. Therefore the strong originality of the research group is highly
esteemed.

Ripple effect: At the beginning of SPring-8 operation, there were only limited number of the facilities and humanresources related to X-ray crystallographic analysis of the protein. Today, however, succeeding protein
purification steps most likely leads to the understanding of the protein crystallization since the quality of domestic
researchers are greatly enhanced by MEXT Protein 3000 Project as well as the important contribution of thisproject group.
Overall evaluation: By pursuing “whole-cell project of T. thermophiles HB8”, this research group significantly
contributed to the development of Japan’s structural biological research and its devotion is highly evaluated.

[REVIEWER 2]
1. Research Object
This group took (1) traditional bottom-up (structural and functional analyses of molecules approaches ofeach molecule by the chemical and biophysical methods and (2) top-down approaches by the omics andwhole-cell imaging methods at the same time to complement the weaknesses of the individual two approaches.
This has the originality and the uniqueness to analyze the whole cell phenomena by using Thermus thermophilus
HB8 as the sample.

2. Research Results
This group has published 154 original papers involving the collaboration with other research groups.
After the interim review, the number of publication increased expectedly. Besides the publication, total 491
protein structures were analyzed and they still maintained the high success rate (over 22%). It showed theevidences that their protein preparation and other technologies must be established. Regarding unknown
functional proteins, the group could predict functions of 60% of total unknown functional proteins. It makes asense for the whole cell project using Thermus thermophilus HB8. Because these proteins exist commonly invarious mammalian cells not limited in Thermus thermophilus HB8. In the interim review, especially two

technologies are very impressive. One is overproduction by the Mistic (membrane-integrating sequence fortranslation of integral membrane protein constructs) tag of Bacillus subtilis to their N-terminals. Twelvetransmembrane proteins were tested for overproduction by the Mistic system, and eight of them were successfullyexpressed. I agree that he success rate of about 65% will be sufficient for the future membrane-protein project forstructural and functional analysis. Another is the technology development for using of XFEL.

Regadring XFEL

method, this group has the obligation for the development of some technologies. Although I guess that there
isn’t enough time to establish new technology, it still didn’t get the proof yet.

3. Management of the Research GroupAfter interim review, this group spread the collaborations with many researchers at RIKEN, universities,institutes, and companies.

I think their efforts for some collaboration absolutely reflected to the results.

4. Comprehensive Review
i.

Construction of resources for whole-cell analysis of a model organism

This group constructed the resources for structural and functional genomics using by using Thermus thermophilus
HB8 as the sample. It achieved to establish some important technologies, especially for the protein expression andpurification.

ii.

Research and development of functional discovery of new proteins (genes)

This group constructed the platforms for top-down approaches, which includes transcriptome and proteome
analyses. By using these platforms, they found new functions of important proteins (genes). Newly identified
proteins must be important for interpreting the whole-cell imaging. In the aspect of developing for functionalgenomics, it is still under developing. The prediction rate of unknown-functional proteins would be moreincreasing by using the informatics involving the experimental data of interactions between various chemicals andunknown proteins.

iii.

Research and development of bio-molecular imaging methods for XFELI think this technology is under development and didn’t reach sufficient level yet.
I hope that these technologies will be developing to the common technologies by this clue in future...

[REVIEWER 3]
This project so called, “Whole-cell Project” launched to understand the ongoing biological life phenomenon ofa cell at atomic level. In order to pursue the project, the integration of two way approaches involves a bottom-up
approach to the protein analysis from the molecular-level to the upper macro level and a top-down approach fromthe cellular-level to the lower micro level. The model organism selected is an extremely thermophilic bacterium,Thermus thermophilus HB8.

1)

Research goal setting:On the way to develop for structural biological research, a deeper understanding of the life phenomenon have

been received attention from not only researching at the protein level but also targeting the upper level of theproteins’ 3D structural and functional research. As part of the attention, this project promotes a way to approach
the research from the molecule level to the cellular level uniformly by bring the whole-cell into a view at once.
The target setting is positively aggressive and disciplinary implication is remarkably strong.

2)

Research achievements:

T. thermophilus HB8 has about 2200 genes (proteins) and or 90% (2000 genes) of them have been successfully
overproduced by using their protein-production system. And establishment of the foundation for the structural andfunctional research is highly evaluated. Furthermore, the approximately 500 protein structures were finalized bypurifying and crystalizing the expressed proteins.

Also there were 500 proteins out of 2,200 which haveunknown functions, and the functions of the 140 out of the 500 were specified.
As the bottom-up approach to the protein analysis from the level of protein, the structures of 500 proteins wereconcluded as the result of devoting large efforts to the determination of the large amount of protein structures.
About the proteins with unknown functions, since approximately 70% of the proteins are predictable to find theway how protein’s main-chain is folded, 80% of the main-chain structures were identified to analyze in the wholeproject. This structural analysis will introduce a new platform which is usable for the upper level of system
biology.
The proteins with identified structures can be categorized into specific functional groups. The transcription,translation, and DNA modification of these functional groups were also analyzed by the teams of Functomics
Biology I Research and Functomics Biology II Research so that these three functional groups lead to the structuraland functional research outcomes.

3)

Research group management:

By holding symposium, the group actively made effort to research outcome presentations and interexchange
among researchers.

4)

Overall (comprehensive) evaluation:

Increasing research quantity at the level of protein compound is succeeded so that it seems that there is anachievement of positive results. Especially Protein 3000 Project showed the successful results by increasing thesuccess rate for this structural prediction. However, if I have to point out the issue of this project with expectation,
the suggestions are as follows.
It is hard to say that the development of analyzing each protein structures can obtains a better
understanding of life phenomena in the upper level of the protein structures. Because it seems that this project
only focused on increasing the large quantity of each specific protein structure. Despite attempting to understandthe life phenomena widely and deeply by the extension of this project, it has been unsuccessfully performed. After
Protein 3000, the project should have only focused on a strategy for the particular functions to understand more.
The structural research at the early stage also should have shifted the direction of the mission into complex
proteins and membrane proteins which are essential to understand the life phenomena. By doing so, we could
have found further progress of the structural research related to the complex proteins which are necessary tocomprehend the functions.
In the research of the bottom up approach (the lower micro level from the cellular level), system biology
research with the advantage of the protein structures would be possibly conducted. Moreover the possibility
should obviously come true, however, within this size of the project group absolutely cannot help for pursuing theunderstanding.
Making continuous effort of analyzing T. thermophiles HB8 has been valuable assets. Therefore as theresearch using SACLA, the effective use of the analysis should be made. In this case, the SACLA should be used
not only for technical evaluation of bio-molecular imaging but also operated by indicating the clarified purposeof biological significance.

[REVIEWER 4]
Synchrotron Radiation System Biology Research Group is based on the structurome project as structural genome
project by using extremely thermophilic organisms. On the basis of the group results, for seven years from April
2006, conducting the comprehensive functional genome research and the functional analysis of individual proteins
the group aims to enable the simulation of whole biological process. As the framework, “Functomics Integration
Research Team” led by group leader Kuramitsu entirely coordinates the whole project to provide a pipeline asprotein productions to protein structural analysis, at the same time, on the basis of platform technical development
for the functional genome research mRNA, protein expression, and metabolic dynamics, furthermore, electron
microscope tomography and x-ray fluorescent imaging were tentatively introduced. It is interesting that theplasmids or databases as the resources are provided to the community. Other two teams conduct structuralfunctional analysis by paying attention to the different sub-systems among extremely thermophilic organisms.
Group of Shinkai mainly focuses on the transcription, and group of Bessho focuses on transcription, translation,replication, and oxidative stress response. Enabling the simulation of all biological process in cells is one of theultimate goals of the life science and the research of this group will make a significant contribution to scientificadvance. As of October, 2012, despite there were members of eight including the team leaders, it is significantly
notable that 157 of original papers were published for seven years and 125 of new structures were registered toprotein databank. Although it is disappointing not to see the goal of understanding whole cells to confirm thepromising future success at this time, there are many outcomes by analyzing individual protein structure and its
function. This result is obvious by reading the newspaper article per attached so that it made a great impact on
the public. As the summary, this project could not accomplish the initial goal perfectly however could create thesignificant results academically so that it succeeded in high level.

[REVIEWER 5]
1. Goal setting：By revealing the three-dimensional all protein structures contained in an extremely thermophilicmodel organism, Thermus thermophilus HB, the goal setting of this group had high-nobility with originality
by understanding the life phenomenon in a cell, from system biological point of view and also provided
academically significant information. Nowadays, the goal of this research is just a standard concept however
when the research group launched, the goal seemed not reachable at all. However, over the short time period,
the group gradually enabled to give a new insight into the life science of the next generation to lead the worldresearch so that the contribution of the group is quite large.
2. Research achievements：The group aimed to understand the functions of a cell by going through the three
dimensional structures and functions of all the proteins in the cell, the outcomes acquired by the highoriginality research goal has a quite high originality as well. By the research results, 154 of the original paper
publications were released on the internationally recognized magazines for seven years so that academic
significance was also great. The information related to the genes and well-constructed overexpress protein
plasmids which distributed to external researchers were enormously valuable as a way to promote proteinscience as well as life science research. Also the research outcomes that have been achieved were went into
headlines and widely spread out through symposiums, so that the achievements had a profound effect onsociety.
3. Research group management：This group is consisted of three teams, and under a team leader each team isconsisted of researchers, PhD researchers, and technical staff, so that it appears that the management wassmoothly practiced. Also a large amount of visiting researchers and graduate students were affiliated, so that it
shows the active research exchanges were established at annual research conference in where the big numberof researchers assembled at once.
4. Overall evaluation：The research contribution can be highly evaluated that a series of system was createdgenomic analysis, protein expression related construction, protein overexpression, purification, crystallization,
diffraction data collection, structural calculation, and functional analysis, also 490 structural proteomics
analysis was completed for seven years. Furthermore, it is also profoundly appreciated that a great mark wasleft for human development and social contribution by accepting visiting researchers and holding researchmeeting. Therefore this project group surely played a remarkable role in the field of structural proteomics.

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