平成 25 年 3 月
構造生理学研究グループ 最終レビュー報告
Structural Physiology 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：
Prof. Masayoshi Nakasako / 中迫雅由
(Faculty of Science and Technology, Keio University)
Dr. Shunsuke Ishii / 石井 俊輔(Molecular Genetics Laboratory, RIKEN Advanced Science Institute (ASI) )Dr. Kaoru Mitsuoka / 光岡 薫
(Protein Structural Information Analysis Team, Biomedicinal Information Research Center,National Institute ofAdvanced Industrial Science and Technology (AIST))
Dr. Kazuhiro Oiwa / 大岩 和弘(Advanced ICT Research Institute, National Institute of Information and Communications Technology (NICT))
評価結果の概要 General comments：[REVIEWER 1]
The Structural Physiology Research Group with aim to understand the atomic to cell scale level
physiologically, from the structural point of view, a regime is created by 4 research teams with different methods
to deal with the research project in a complementary style. And the regime addresses the basic technology
development to help structural understanding and the application of the research. Research method using highintensity X-ray source from SACLA and SPring-8, and the R&D which is complementally usable for the method
are academically valuable to fill in the resolution gap between the best (ideal) and the actual situation of structuralbiology, and also the technologies produced by the research activities and applied to biological living samples are
physiologically important for proteins associated with synapses and sensory perceptions, as well as cytoskeleton.
Bio-multisome Research Team, by using electron microscopes, addressed the research of filling the resolution gap
between light microscopes and x-ray microscopes (used in x-ray crystal structural analysis). Especially thedevelopment of genetically encoded tags enabling protein detection by electron microscopes and the applicationof the encoded tags succeeded to observe the protein localization to synapse. Hereafter much further improvement
is required for the general use of the microscopes, however the approach of the field of microscope R&D which is1

important for the electron microscopes is highly evaluated. Also currently this team is working on a correlated
light and electron microscope, and also it succeeds to identify the localization of acetylcholine receptors so that
we can expect further future development. Such R&D will be gradually more significant to make the connection
between atomic models created by the x-ray crystal structural analysis and the functions of the actual cells.
Three dimensional microscopy research team made efforts on two dimensional crystallization of membraneproteins and the crystallization was applied to the crystal structure analysis by electron beam, and the team wasalso collaborated with Molecular Signaling Research Team to address the three dimensional crystallization. About
the electron beam analysis of crystal structure, the three dimensional conformational transporter of the AE1 (anionexchanger 1) was determined and also the three dimensional crystals of oxalate transporters are obtained so thatthe improvement of the crystallization are expected.
Molecular Signaling Research Team with an aim to understand the mechanism of the sensor molecularfunctions, the mechanism related to the protein structural determination and the functional analysis of the sensor
molecules are addressed.
About acetylcholine-binding proteins which are homologous to the substrate-binding sites of acetylcholine
receptors, complex molecule structures binding to some pesticides were determined. Such detailed structural
analysis seems significant in order to develop the specific medical substances. Also heterodimers of T1T2 andT1T3 as gustatory receptors were structurally analyzed and also TRP channel as mechanosensitive channel wereanalyzed using NMR. For the protein crystallization screening, new techniques such as native electrophoresis andother methods were developed. Therefore future use of these methods is promising.
X-ray Structural Analysis Research Team structurally analyzed for F-actins and the F-actin capping protein
complexes in diverse ways. First, high-resolution structure was determined by using fiber diffraction and mutantactins were analyzed. And to understand the motility, inelastic scattering measurements and molecular dynamics
calculations were introduced.

Also collaborative research in actin-related protein complex was conducted.

Through these wide ranges of analysis, highly complementary results (figures) were obtained so that other fibril
proteins such as amyloid fibrils are promisingly applied.
Some other collaboration researches within some groups are also in progress and all teams are engaged withsome kinds of collaborative research so that it seems that the management system interactively activates entire
research. Also each team has a clear and challenging research targets and these research are conducted in orderto understand them. Although the research methods are widely varied, since two teams are mainly engaged withSPring-8 (synchrotron radiation) and other two teams are engaged with complementary methods (as using electronmicroscopes) and both ways are appropriately balanced. Moreover each group sets a challenging goal so that thefuture development will be positively expected.

[REVIEWER 2]
1. Research Subject
From the self-evaluation report, it is difficult to determine the main subject of this project. Summarizing
the research activity of this project including the published peer reviewed papers, the focus seems to be a study ofthe mechanisms for cellular motility and communication at the molecular level. The research teams worked as2

users employing various existing imaging techniques developed in the field of diffraction physics rather than asinnovators seeking to develop original techniques for SPring-8, SACLA and electron microscopy. Thus, theresearch carried out in the seven-year project seems to center on sample preparation to allow visualization ofcellular and molecular structures.

2. Scientific Results
This seven-year project, which spent a grant of approximately 750 million yen, is large enough to raisehigh expectations for important contributions to the fields of biological and biophysical science. Below, I tried tobriefly summarize the original achievements of the four research teams. During the past 7 years, the scientificresults from this project probably have little social impact.
The bio-multisome research team seemed to focus on sample preparation techniques using
commercially available electron microscopes. The approach is important to develop methods using electron
microscopy in biological science as well as done in some groups of national universities. As a result, this grouppublished four papers closely related to the main theme. The research that was carried out mostly within the groupis highly evaluated. Collaboration with research groups outside RIKEN is an important prerequisite for this teamto publish papers in various fields.
Two of the research teams studied the structures of membrane proteins. The Three-dimensional
Microscopy Research Team focused on the human erythrocyte anion exchanger 1 at low resolution, and publishedseveral papers in collaboration with some experts of electron microscopy. The Molecular Signaling Research
Team received 161.4 million yen during the period from 2007 to 2012, and published probably two papers
reporting the original sample preparation techniques concerning the project. The other publication are better to becategorized as not main but side projects of this team. The small number of original papers from these two
research teams indicates the difficulties and risks in structural biology studies of membrane proteins. The
researchers would reveal many three-dimensional structures of membrane proteins at high resolution in the near
future based on the currently unpublished research results.
The X-ray Structural Analysis Research Team focused their research on the structure-function of actin.
The scientific significance of the research subject is well known in biophysics and biology. This team worked wellunder the direction of Professor Maeda of Nagoya University and proposed a structural model of actin filament
through very simple calculation of the fiber diffraction pattern obtained at SPring-8. In addition, by providing
samples to the neutron scattering group at Tokai Institute of the Japan Atomic Energy Agency, this teamcontributed to measure the 'ensemble-averaged' structural fluctuation of actin filament. Judging from the numberand quality of the peer-reviewed publications, this team was the most active among the four teams, despite
receiving the smallest amount of grant money during the seven years.
Operations of the SACLA XFEL facility for public users started in 2012. The project leader emphasizes
that their novel techniques in electron microscopy are suitable for XFEL experiments using biological samples.
However, it is very surprising and unfortunate for RIKEN that no one in four research teams used SACLA during
this project.

33. Management
This research group seems to be a simple assembly of four research teams of biophysicists, who studied
different targets using different experimental techniques, although the group leader emphasized his efforts toorganize scientifically the four teams, that were directed relatively young scientists without experiences inscientific researches and laboratory management. People in the biophysics field would sympathize with the groupleader for his struggles to coordinate this disorganized group.

4. Summary
In summary, the research project contributed to structural biology of actin based on the previous
research project conducted by Professor Maeda. The other three teams also carried out nice researches forstructural studies of cellular structures and membrane proteins. The multi-some research must be quite difficult,judging from the small number of peer reviewed publications and invited lectures, which are closely related to the7-years research project. Thus, it is very difficult to say simply this project, which spent the large grant ofapproximately 750 million yens, ends in failure except for a model to tentatively discuss the structure-function
relation of actin. And, it is now a total mystery why managers of Harima RIKEN selected this project 8 years ago.

[REVIEWER 3]
The Structural Physiology Research Group, based on the seven-year-plan from April 2006, has been promoting
the research development related to the structures of the membrane proteins and cytoskeletal proteins related tocellular information transmitting as well as the physiological functions of the proteins. This group consists of fourteams and comprehensively addresses to combine three analysis methods of X-ray spectroscopy (structuralanalysis), electron microscopy, and light microscopy. Centering the analysis information transmission in thesynapse, the four teams aimed to create the synergy effect by analyzing the various membrane proteins related tothe information transmission and the three dimensional dynamic physiological function of the complexes.

Under the management of respecting the each distinctive research team, the research activity of this researchgroup launched by collecting bright futures young researchers produced excellent outputs individually. On theother hand, since the direction of the whole group was not positive going, the enhancement of the collaborative
research among the teams produced the synergy effect in a limited way. Therefore there was no remarkable
creation of technology and academic finding as representing outcomes of RSC. The research project of RIKEN is
expected to create innovative technologies and academic outcomes by utilizing the cutting edge facilities. From
this point of view, the top management should be required. Also this group set the important and difficult themes
to challenge aggressively, however the human power resource was not enough and some researchers transferred
out in the middle of the project. As the result, the quantity of the research papers from the group was not sufficientso that the “manuscript in preparation” of papers should be in hurry.

Considering how to build the framework toaddress the challenging project, the researchers should find a room for the improvement. Good things to note isthat many of the team leaders and researchers who were remarkably active, found independent positions inuniversities and started their own research activities therefore the purpose of educating the next generation leaders4

for the structural biology was accomplished.

[REVIEWER 4]
1. Research Objectives: novelty, specific significanceThe Structural Physiology Research Group aims to study a dynamic physiology from three dimensional
structure analyses of proteins and complexes, which are important for signaling, by an X-ray crystallographic
analysis and an electron microscopy. Although the targets of this research project are diverse, this group sharescommon sense of research analysis, and their research objectives are appropriate.

2. Research Results: Originality, specific significance social impact
All of four teams obtained superior results. For instance, 1) Bio-multisome Research Team developedmultiple novel techniques for observation by electron microscopy, such as STEM (scanning transmission electronmicroscope) tomography. 2) Three-dimensional Microscopy Research Team succeeded to obtain low-resolution
structure of AE1, the most abundant membrane protein in human erythrocyte. 3) Molecular Signaling Research
Team determined the crystal structure of LeuT, the highly-stable NSS (neurotransmitter sodium symporters)
homolog, in two different functional state. 4) X-ray Structural Analysis Research Team clarified the nature of theglobular- to fibrous-actin transition by creating a model of F-actin using X-ray fibre diffraction intensities. Eachresults has high quality of science, and their social ripple effect is very strong.

3. Management of the Laboratory: Laboratory member composition, researchers interaction etc.
Many talented scientists from different fields gather and form this unique group. Advantage of thisgroup seems to be a respect of “bottom-up science”, and most of research subjects appeared to be derived from theresearcher side. This may be due to a direction of Dr. Miyazawa, the leader of this group, who respects theindividuality of each team leaders. One of the criticisms at the Midterm Review of this group in 2010 was a lackof cooperative works between four research teams. However, some high-level studies were performed
cooperatively by multiple teams, and this criticism has been now improved.

4. Overall assessment
This group succeeded to obtain unique and superior results, each of which has high quality of science.
This is mainly due to excellent talented scientists are gathered from various fields, and also due to the “bottom-updirection” by the leader of this group.

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