“ Molecular Movies” International Symposium 2023
高速分子動画 国際シンポジウム 2023 要旨集

Prog ram_プログラム
1.

Oral Session_⼝頭セッション2.

Short-Talk Session_ショートトークセッション

3.

Project Meeting_領域会議

4.

Poster Session_ポスターセッション

Abstract_要旨
5.

Oral Session_⼝頭セッション

6.

Short-Talk Session_ショートトークセッション

Organizer
Grant-in-Aid for Scientific Research on Innovative Areas “Non-equilibrium-state
molecular movies and their applications (Molecular Movies)”
Supported by
The NOVARTIS Foundation (Japan) for the Promotion of Science
Kato Memorial Bioscience Foundation
Inoue Foundation for Science
The Kyoto University Foundation

“Molecular Movies” international symposium 2023

1. Oral Session Program
Thu. 30th Novembe r
Speaker

Affil iation

Title

C hair

12:00-13:00

Set up posters

13:10-13:20

So Iwata

Kyoto
University

Opening Remarks

13:20-13:55

ShigekiKiyonaka

NagoyaUniversity

Molecular- targeted c hemogenetic s, anext- generation tec hnique formanipulating cell- sur face receptors

Nagasawa

13:55-14:30

Yoshitaka
BesshoTime- res olved c rystallogra phy o fultrafast light- driven DNA repa ir byphotolyasesPark

14:30-15:05

Jiangyun
Wang

Molecule movies enabled by genetic
code expansion (Onli ne)

Kiyonaka

RIKEN SPring-8
Center/University ofTokyo
ChineseAcademy of
Sciences

Break ( 15:05-15:15)
15:15-15:50

Osamu
Miyashita

RIKEN R-CCS

15:50-16:25

AnaNicoleta
BondarUniversity of
Bucharest

16:25-17:00

Gerhard
HummerMax Planck
Institute ofBiophysics

Integrative/Hybrid ModelingApproac hes for Dyn amic Struc tura lBiology
Water- mediated hydrogen- bond
networks fo r signal rel ay in G Protein
C oupled Receptors (On line)
Molecular simulatio ns in the e ra o f AI
and exasc ale c omputing ( Online )ShinodaMiyashita

Shoji

Break ( 17:00-17:10)

17:10-17:45

Takeshi
Murakawa

Osaka Medical
andPharmaceutical
University

17:45-18:20

Valérie
PanneelsPaul Scherrer
Institut

18:20-18:55

Christopher
Schofield

University of
Oxford

18:55-19:00

So Iwata

Kyoto
University

Molecular movie o f the catalytic
reac tion o f bac terial c opper am ine
oxidase conducted by mix- and- inj ec t
serial femtosecond X- ray
crystallograp hy
Ultrafast structural changes di rect thefirst mo lecular events of vis ion(Online )Time- res olved studies on themechanisms o f ox ygenases and related
enzymes (Onl ine)
Closing Remarks

Dinner（19:30-21:00）
21:00-22:30

Poster Session

22:30-

Remove posters
2

KimuraNango

Nagano“Molecular Movies” international symposium 2023

2. Short-Talk Session Program
stFri. 1 December
Room

Event hall

403

404

A01_Structural biology

A01_Structural biology

405

B01_Molecular Movie Platform

C01_Computational Chemistry

Design＆C01_Spectroscopy

Chair

Iwata

Park

Yamamoto／ Kubo

Miyashita

9:00-9:10

Masahiro Fukuda

Michihiro Suga

Hiroaki Matsuura

Kiyoshi Yagi

9:10-9:20

Toshiaki Hosaka

Atsuhiro Shimada

Akihiro Suzuki

Mitsuo Shoji

9:20-9:30

Ritsu Mizudori

Junpei Yamamoto

Tetsunari Kimura

Duy Phuoc Tran

9:30-9:40

Kota Katayama

Yasufumi Umena

Yosuke Mizuno

Akio Kitao

9:40-9:50-

Sam-Yong Park

Yuji Furutani

Ayori Mitsutake-

Tetsuro Katayama (Online) -9:50-10:00 A01_Chemical biology
Chair

Kiyonaka

A01_Structural biology

C01_Computational Chemistry

NaganoShoji

10:00-10:10 Takuya Terai

Toru Nakatsu

Shigehiko Hayashi (Online)

10:10-10:20 Takushi Shimomura

Takehiko Tosha

Junichi Ono

10:20-10:30 Tomohiro Doura

Eiichi Mizohata

Sriram Srinivasa Raghavan

10:30-10:40 Tsuyoshi Araya

Shingo Nagano

Keiko Shinoda

Coffee Break & Photo Shoot（10:40-11:15)

3.

新 学 術 領 域 「 ⾼ 速 分 ⼦ 動 画 」 領 域 会 議 プ ロ グ ラ ム (Closed)
12 月 1 日 （ 金 ）講演者

11:1511:21
11:2111:27

所属

タイトル

岩田 想

京都大

はじめに／構造生物の結果

清中 茂樹

名古屋
大

高速分子動画撮像に向けたケミカル光制御技術の開発

11:2711:33

南後 恵理子

東北大

11:3311:39

久保 稔

11:3911:45
11:4512:00

宮下 治

時分割実験のための多様な反応誘起シ
ステムの開発時間分解分光で観るタンパク質の反応
ダイナミクス
分子シミュレーションによるタンパク
質化学反応ダイナミクスの解明

兵庫県
立大
理化学
研究所

質疑応答・アドバイザーコメント

3

“Molecular Movies” international symposium 2023

4. Poster Session Program
Thu. 30th November / 9:00pm- JST / Lobby
名前所属

ポスタータイトル1

荒谷 剛史

京都大学医学研究科

Crystal structure of photoresponsive ligand bound adenosine A2A
receptor2

安東 智大

京都大学理学研究科

イクオリンの生物発光過程についての理論的研究

3

伊藤 朱音

京都大学医学研究科

アンタゴニスト結合型ヒスタミンH4受容体の立体構造解析

4

井上 始

名古屋大学工学研究科

化学遺伝学的手法を用いたセロトニン5-HT2A受容体に対するAntagonistの親和性制御

5

梅名 泰史

名古屋大学シンクロトロン光
研究センターFast time-resolved structural analysis of light-response inphotosynthetic antenna protein Phycocyanin

6

江尻 智森

京都大学理学研究科化学専攻

アニオンポンプロドプシン NpHR のスイッチング機構解明に向けた理
論的研究

7

戎 唯良汰

東京大学理学系研究科

Cryo-EM structure analysis of Schizorhodopsins from mesophilicarchaea8

王 雨竹

東京大学大学院理学系研究科

藍色光を受容するチャネルロドプシンの構造及び機能解析

9

小野 純一

Waseda University

Hybrid in silico drug study based on quantum molecular dynamics with
virtual screening and docking

10 片山

耕大

名工大院工

Identifying functional hotspot residues for activation in M2 muscarinic
receptor (M2R)

11 北尾

彰朗

東京工業大学生命理工学院

Molecular mechanisms of structure support and smooth rotation in thebushing/rod complex of bacterial flagella

12 木村

哲就

神戸大学大学院理学研究科

Stepwise calcium binding in photoluminescence protein; aequorin,
revealed by the time-resolved spectroscopy

13 近藤

匠

名古屋大学工学研究科

作用薬の機能転換を利用した代謝型グルタミン酸受容体1の活性制御

渉

東京大学理学系研究科

Cryo-EM sructure of the zeaxanthin-xanthorhodopsin complex

15 島田

敦広

岐阜大学応用生物科学部

XESとXRDの同時測定法を用いた、シトクロム酸化酵素の反応機構解
明の試み

16 下村

拓史

生理学研究所 神経機能素子
研究部門

Characterization of a light-gated K+ channel generated by introducing
an photo-isomerizing unnatural amino acid

17 庄司

光男

筑波大CCS

Overview of our research collaborations performed in the researcharea of “molecular movies”

18 Duy Phuoc Tran

東京工業大学・生命理工学院

How the mGLU1 receptor dimer be activated: a molecular dynamics
study

19 菅

岡山大学異分野基礎科学研究所

Real-Time Structural Changes during the Light-induced Water Oxidationof Photosystem II Caught by Time-Resolved Crystallography

名古屋大学工学研究科

光薬理学によるアデノシンA2A受容体のサブタイプ選択的な活性化

RIKEN

ResiDEM: Analytical Tool for Isomorphous Difference Electron Density
Maps Utilizing Dynamic Residue Identification via Density Clustering.

14 志甫谷

20 鈴木

倫寛

21

啓文

Sriram Srinivasa
Raghavan

22 鈴木

明大

北海道大学電子科学研究所

Low background graphene sample holder for XFEL-based
crystallography and imaging

23 田口

真彦

東北大学多元物質科学研究所

Understanding of protein functional expression using hybrid QM/MM
free energy method

4

“Molecular Movies” international symposium 2023

名前所属

ポスタータイトル

24 田中

達基

東京大学理学系研究科

Structural basis for the highly sensitive channelrhodopsin GtCCR4

25 永澤

秀子

岐阜薬科大学

生理活性作用を有するデカリン生成のための化学的および酵素的分子内Diels-Alder環化付加反応の立体制御機構の解明

26 Nipawan Nuemket

JASRI

Towards Elucidating the Structural Dynamics of Animal and Microbial
Rhodopsin using Time-Resolved Crystallography at XFEL and
Synchrotron

27 Basudev Maity

Tokyo Institute of Technology

Observation of a CO release reaction within protein crystal.

28 長谷川

JASRI

SPring-8 BL41XUにおける時分割構造解析環境の構築

高輝度光科学研究センター

Non-cryogenic X-ray crystallography under various ambient conditions
using the humid air and glue-coating (HAG) method

名古屋大学工学研究科

ループ工学に基づいたドーパミン受容体の活性制御

29 馬場

和也

30 原

清喜
隆史

31 日野

智也

鳥取大学工学研究科

プレートスキャンシステムを用いたTRPV3の新規微結晶化条件の探索

32 福田

昌弘

東京大学 先進科学研究機構

Structural and functional analyses of pump-like channelrhodopsins

33 藤原

孝彰

東北大学多元物質科学研究所

テルペン環化酵素CotB2の室温結晶構造解析

34 保坂

俊彰

理研 BDR

Synthesis of photocaged L-tyrosine containing proteins through an
Escherichia coli cell-free protein synthesis system for time-resolved
structural analysis

35 松浦

滉明

理化学研究所 放射光科学研
究センター

Development of an in-vacuum diffractometer for protein microcrystallography

36 松岡

佑真

名古屋大学工学研究科

細胞外ループに着目したケモジェネティクスによるアデノシンA2A受
容体の自在な制御

37 水鳥律

名古屋工業大学大学院 工学専攻

Crystal structure of V2HeR3, a Viral heliorhodopsin transporting proton

38 水野

秀昭

Department of Chemistry, KU
LeuvenDecarboxylation via two-photon absorption process results in efficient
LSSmOrange photoconversion

39 水野

陽介

名古屋工業大学

Structural features of Lumi intermediate on a primate blue-sensitive
visual pigment revealed by FTIR

40 光武

亜代理

明治大学理工学部物理学科

Analysis of protein simulations using relaxation mode analysis and 3DRISM theory.

41 宮崎

育実

東京大学大学院理学系研究科

A caged lactate for light-induced biosensor activation

42 八木

清

理研CPR

非断熱QM/MM分子動力学計算法の 開発と応用

43 山元

淳平

大阪大学大学院基礎工学研究科Time-resolved serial femtosecond crystallography of light-induced
structural changes of a bifunctional cryptochrome駿

Department of Physics, Schoolof Science and Technology,
Meiji University / Department ofStructural Biology, School of
Medicine, Stanford University

Structural and Computational Insight into Dynamics and Intermediate
State in Activation of Orexin 2 Receptor

45 Luo Fangjia

JASRI

Time-resolved structure analysis enhanced by mixing and improved
liquid injectors

46 長谷川鳥取大学工学研究科

立体選択的なDiels-Alder反応を行う酵素の生成物結合型結晶構造とその反応過程の分子動画撮影に向けた取り組み

44 横井

颯人

2023.11.29

5

“Molecular Movies” international symposium 2023

5. Oral Session Abstract
Thu. 30th November / 1:10pm- 7:00pm JST / Event Hall

Molecular-targeted chemogenetics, a next-generation technique formanipulating cell-surface receptors

Prof. Shigeki Kiyonaka (Nagoya University)
Cell surface receptors transmit extracellular information into cells. Spatiotemporal
regulation of receptor signaling is crucial for cellular functions, and dysregulation of
signaling causes various diseases. Thus, it is highly desired to control receptor functions
with high spatiotemporal resolution. Conventionally, genetic engineering or chemical
ligands have been utilized to control the receptor function. As the alternative,
chemogenetics has been recently proposed, in which target proteins are geneticallyengineered to interact with a designed chemical partner with high selectivity. The
engineered receptor can dissect the function of one receptor member among a highly
homologous receptor family in a cell-specific manner. In most cases, engineered receptors
can be activated by designer ligand instead of the endogenous ligand. However, neither
cellular responses nor the timing of activation may reflect the physiological responses.
To overcome the shortcomings of the current techniques, we have recently proposed"molecular-targeted chemogenetics" which allows the cell-type specific control of target
endogenous receptors without affecting original receptor function. In this talk, I will
present recent progress about “molecular-targeted chemogenetics” and its applicability
for molecular movie studies.

Time-resolved crystallography of ultrafast light-driven DNA repair byphotolyases

Dr. Yoshitaka Besho (RIKEN SPring-8 Center/ University of Tokyo)
DNA photolyases are flavoenzymes that repair UV-induced DNA pyrimidine dimer
damage, such as cyclobutane pyrimidine dimers (CPD). Blue light activates the enzyme
through the photoreduction of its flavin adenine dinucleotide (FAD) cofactor. In theprocess of photoactivation, two light-triggered single-electron photoreduction steps
convert the oxidized chromophore (FADox), via the radical semiquinone state (FAD•-

6

“Molecular Movies” international symposium 2023

and its subsequently protonated form FADH•), to the reduced hydroquinone state(FADH-). To clarify the photoreduction mechanism of photolyase, we used thefemtosecond pulses of the SACLA X-ray Free-Electron Laser (XFEL) as a probe. The
damage-free crystal structure of Methanosarcina mazei CPD photolyase revealed theexact arrangement of the water molecule clusters adjacent to the U-shaped FAD cofactor.
By using time-resolved crystallography, we found the reaction intermediates in the lightdependent reduction of photolyase, at atomic resolution. We could observe timedependent buckling and twisting in the isoalloxazine ring of the FAD cofactor in different
redox states. In addition, our results uncovered the molecular repair mechanism for
CPD-containing DNA. Our 3D molecular movies showed how the protein environmentof redox cofactors organizes multiple electron and proton transfer events in an orderedfashion. At this symposium, we will report our latest results.

Molecule movies enabled by genetic code expansion

Prof. Jiangyun Wang (Chinese Academy of Sciences)
One of the primary objectives in chemistry research is to observe atomic motions during
reactions in real time. Although X-ray free-electron lasers (XFELs) have facilitated thecaptureofreactionintermediates

using

time-resolved

serialfemtosecond

crystallography (TR-SFX), only a few natural photoactive proteins have been
investigated using this method, mostly due to the lack of suitable phototriggers. Here we
report the genetic encoding of a xanthone amino acid (FXO), as an efficient phototrigger,
into a rationally designed human liver fatty-acid binding protein mutant (termed XOM),
which undergoes photo-induced C–H bond transformation with high selectivity andquantum efficiency. We solved the structures of XOM before and 10–300 ns after flash
illumination, at 1.55–1.70

Å resolutions, and captured the elusive excited-stateintermediates responsible for precise C–H bond activation. We expect that most redox
enzymes can now be investigated by TR-SFX, using our method, to reveal reaction
intermediates key for their efficiency and selectivity.

7

“Molecular Movies” international symposium 2023

Integrative/Hybrid Modeling Approaches for Dynamic Structural Biology

Dr. Osamu Miyashita (RIKEN R-CCS)
Understanding the complex mechanisms that govern protein functions depends on
knowledge of their structures and dynamics. While experimental techniques are
essential for such studies, computational methods are equally important. Experimental
data are limited in resolution and require computer simulations to obtain a detailed
picture of protein dynamics. Emerging technologies that generate large and complex
data sets also pose challenges. On the other hand, computer simulations are limited inaccuracy due to computational cost. Therefore, the integration of computational
simulations and experimental data is essential.
In this presentation, we discuss two integrative modeling approaches. One example isthe use of molecular dynamics (MD) simulations to interpret time-resolved serial
femtosecond crystallography (TR-SFX) data. This method supports the construction of
structural models from complex TR-SFX data by combining structure sampling based on
MD and consistency measurements between models and data. The second example
concerns the analysis of cryo-electron microscopy image datasets. Here, our goal is toobtain a complete conformational ensemble of the biomolecules in the sample byintegrating computational simulations with image datasets, leading to a better
understanding of protein dynamics.
The integration of computational methods and experimental data is a powerful tool in
the elucidation of protein structure and dynamics. Through these illustrative integrativemodeling approaches, we hope to demonstrate their potential and discuss further
extensions in the field of protein research.

Water-mediated hydrogen-bond networks for signal relay in G Protein
Coupled Receptors

Prof. Ana-Nicoleta Bondar (University of Bucharest)
G Protein Coupled Receptors (GPCRs) are seven-helical membrane proteins thatmediate cell-signaling paths used by eukaryotic cells to communicate with their
extracellular environment. Upon binding of an extracellular agonist ligand the GPCR
changes conformation, becomes activated, binds and activates a cytoplasmic cytoplasmic
G protein partner; this starts a cellular signaling cascade that ultimately leads to a8

“Molecular Movies” international symposium 2023

physiological response. We seek to understand the general principles of how GPCRs relay
across the membrane changes in structure and fynamics. To this aim, we develop graphbased algorithms and graphical user interfaces that are particularly suitable to dissect
dynamic water-mediated hydrogen-bond networks. We apply these algorithms todatasets of static GPCR structures, and to atomistic molecular dynamics simulations of
GPCRs. The talk will present the principles of these algorithms, and focus on
applications on GPCRs for which protonation states of the receptor and/or ligand areassential for function. The graph-based analyses identify dynamic water-mediatedhydrogen-bond networks that extend throughout the receptors and relay structural
perturbations across the membrane.

Molecular simulations in the era of AI and exascale computing

Prof. Gerhard Hummer (Max Planck Institute of Biophysics)
Rapid growth in raw computing power and advances in artificial intelligence areushering in a new era in biomolecular modeling and simulation. On the one hand, amassive expansion in aggregate computing allows us to tackle ever larger biomolecularsystems; on the other hand, the development of sophisticated artificial intelligenceframeworks provides critical support for the design, operation, and analysis of thesesimulations. In my presentation, I will showcase our efforts to tackle the triple
challenges of system size, complexity, and time scale. I will highlight our push towards
cell-scale molecular simulations with a focus on the nuclear pore complex scaffold andits FG-nucleoporin permeability barrier. I will also describe our efforts to develop a selflearning AI framework to manage molecular dynamics simulations autonomously, withthe aim to resolve seconds-scale dynamics in microsecond-scale simulations and reveal
the mechanisms of biomolecular self-assembly and function.

Molecular movie of the catalytic reaction of bacterial copper amine oxidase
conducted by mix-and-inject serial femtosecond X-ray crystallography

Dr. Takeshi Murakawa (Osaka Medical and Pharmaceutical University)
In order for enzymes to catalyze chemical reactions effectively and specifically, it isessential to optimize the active site environment at each reaction step, thereby reducing
9

“Molecular Movies” international symposium 2023

activation energy and achieving reaction specificity. Studies based on this perspective
require structural information with high time resolution. The present study aims at
elucidating kinetically and structurally the precise catalytic mechanism of copper amineoxidase from Arthrobacter globiformis (AGAO) using mix-and-inject serial femtosecond
X-ray crystallography (MISC).
The overall catalytic reaction of AGAO is composed of two half-reactions: the first
reductive and the second oxidative half-reactions based on the redox state of a quinone
cofactor, topa quinone (TPQ). The catalytic mechanism of this enzyme proceeds via aping-pong bi-bi mechanism. In this study, AGAO microcrystals were prepared by
combining micro-seeding and batch crystallization methods. MISC was conducted underanaerobic conditions to detect structural changes in the reductive half-reaction. We
obtained several data sets at SACLA with different time delays after mixing the
microcrystal solution of AGAO with the substrate solution. The data with a delay timeof 1.0 s yielded only the structure of the semiquinone intermediate (TPQsq), the final
product of the reductive half-reaction, indicating that the reductive half-reaction
proceeds within 1.0 s in the crystal. In the symposium, we will discuss the catalytic
mechanism of AGAO based on several structural data obtained at each delay time of
MISC.

Ultrafast structural changes direct the first molecular events of vision

Dr. Valérie Panneels (Paul Scherrer Institut)
Matthew Rodrigues1, Thomas Gruhl1, Tobias Weinert1, Schertler group1,2, Nango group3,
Neutze group4, Nogly group2, SwissFEL group5, SACLA group6, Standfuss group1,
Gebhard Schertler1,2, Valérie Panneels1.
1 Division of Biology and Chemistry, Paul Scherrer Institute, 5232 Villigen, Switzerland
2 Department of Biology, ETH Zurich, 5232 Villigen PSI, Switzerland.
3 RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
4 Department of Chemistry and Molecular Biology, University of Gothenburg, Sweden.
5 SwissFEL, Paul Scherrer Institute, 5232 Villigen, Switzerland
6 Japan Synchrotron Radiation Research Institute (JASRI), Hyogo, 679-5198, Japan.

Mammalian rhodopsin is our light receptor for vision. It belongs to the highly druggable10

“Molecular Movies” international symposium 2023

G protein-coupled receptor family. It hosts the retinal chromophore which, like a switch,
isomerizes in less than 200 femtoseconds upon photon absorption. This triggers
sequential intramolecular changes in rhodopsin, initiating the signalling cascade
generating in milliseconds vision events to the brain via the optic nerve. However, theintramolecular events transforming the rhodopsin resting state[1-2] (dark state) into thetransducin-binding activated state[3-5] (Meta II) are not completely understood.
We now experimentally determined the ultrafast changes of native bovine rhodopsin atroom temperature using time-resolved serial femtosecond crystallography, alreadysuccessfully used for the proton pump bacteriorhodopsin[6-7], at SACLA and SwissFEL
X-ray free electron lasers. Thousands of rhodopsin microcrystals grown in the dark aresuccessively injected in the light of a pump laser and probed after various time-delays
using an XFEL. After 1 picosecond, we observe a highly distorted all-trans retinal that
has induced changes in its binding pocket while the excess energy of the absorbed 480
nm-photon dissipates inside rhodopsin through an anisotropic protein breathing motion
towards the extracellular domain. Interestingly, some amino acids known to be key
elements later in the transduction of the signal are involved in the ultrafast changes.

D.O.I.s[1]10.1016/j.jmb.2004.07.044

10.1038/nature09789[2]10.1016/j.jmb.2004.08.090[5]10.1073/pnas.1114089108[3]10.1038/nature09795[4][6]10.1126/science.aah3497[7]10.1126/science.aat0094

Time-resolved studies on the mechanisms of oxygenases and related
enzymesProf. Christopher J. Schofield (University of Oxford)
The lecture will describe time resolved crystallographic studies on the mechanisms of
2-oxoglutarate dependent oxygenases and related enzymes using both conventional
and XFEL methods, as well as supporting data in solution. The results illustrate how
XFEL studies can provide information on dynamic changes during catalysis that are
not readily obtained by any other currently available method.

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“Molecular Movies” international symposium 2023

6. Short-Talk Session Abstract<A01_Structual biology>
Fri. 1st December / 9:00 -9:40am JST / Event HallLight energy conversion mechanism of photosensitive membrane proteins
revealed by cryo-electron microscopy

Masahiro Fukuda (The University of Tokyo)
In nature, a variety of light-sensitive proteins exist and convert light energy into various
types of work. Channelrhodopsins (ChR) are representative photosensitive membraneproteins that can convert light energy into ion transport through cell membranes.
Although ChRs are widely used as an optogenetic tool in fields such as neuroscience,
there are still many unknown points on the molecular mechanism of ChRs. In this talk,the molecular mechanism of recently discovered unique ChRs will be presented.

Synthesis of photocaged L-tyrosine containing proteins through an
Escherichia coli cell-free protein synthesis system for time-resolved
structural analysis

Toshiaki Hosaka (RIKEN Center for Biosystems Dynamics Research)
Genetically encoded caged amino acids can be used to control the dynamics of protein
activities and cellular localization in response to external cues. In this study, we haveestablished a protein preparation system that introduces photocaged Tyr at arbitrary
positions using an Escherichia coli cell-free protein synthesis system. Using this system,
we prepared lysozyme and homoisocitrate dehydrogenase with photocaged Tyr, andcrystallographic study showed that the site-specifically incorporated photocaged Tyr
were degraded to tyrosine by light irradiation on the crystals. Thus, this cell-free protein
synthesis of proteins with the photocaged Tyr could facilitate time-resolved structural
analysis of proteins, including medically important membrane proteins.

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“Molecular Movies” international symposium 2023

Crystal structure of V2HeR3, a Viral heliorhodopsin transporting proton

Ritsu Mizutori (Nagoya Institute of Technology)
Heliorhodopsins (HeRs), a recently discovered rhodopsin family, are widely present inarchaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remained
unknown. In 2022, we reported that a viral HeR from Emiliania huxleyi virus 202(V2HeR3) is a light-activated proton transporter. Comprehensive mutagenesis study
using electrophysiological experiment revealed some amino acids together with theretinal Schiff base forming the proton transporting gate. Here, we show the 2.1 Åresolution structure of V2HeR3. Structural and spectroscopic analyses reveal V2HeR3
specific water-mediated hydrogen bond network in both extracellular and cytoplasmic
sides. These structural elements are believed to enable proton selective transport
functions.

Identifying functional hotspot residues for activation in M2 muscarinic
receptor (M2R)

Kota Katayama (Nagoya Institute of Technology)
The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR)
that serves as a model system for understanding both ligand recognition and activationof GPCR. Here, we identify the mechanisms governing M2R activation flexibility using
vibrational spectroscopy in combination with mutagenesis and organic synthetic
chemistry, focusing on the native agonist acetylcholine. From these studies, we foundthat the exquisite distance between acetylcholine and Asn404, one of the amino acids
that constitute the ligand binding site, is important for M2R activation, and that the
N404Q mutant exhibits partial activity-like conformational changes. Thus, these results
strongly indicate that Asn404 is a hotspot residue in M2R activation.

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“Molecular Movies” international symposium 2023

<A01_Chemical biology>
Fri. 1st December / 10:00 -10:40am JST / Event Hall

Next Generation Biosensors Enabled by High-speed Visualization of
Dynamic Mechanisms

Takuya Terai (The University of Tokyo)
Fluorescent protein (FP)-based biosensors are powerful tools in biology research, but
only a few biosensors have excellent performance. A major limitation for theirdevelopment is the lack of understanding of the mechanisms by which they operate. To
address this issue, we are trying to capture a Molecular Movie of a crystallized FP
biosensor that responds to a ligand released by light illumination. We have synthesized
photocaged lactate and validated its performance. We have got grown crystals of lactate
biosensors in their apo states, though the quality may need further improvement The
latest progress will be described in the presentation.

A light-gated K+ channel generated by introduction of a photo-isomerizing
unnatural amino acid

Takushi Shimomura (National Institute for Physiological Sciences)
The introduction of genetically encoded unnatural amino acids (UAAs) allows us to
confer a variety of functions on target proteins. We introduced phenylalanineazobenzene (Pab), a photo-isomerizing UAA, into KcsA, a pH-dependent K+ channel.
Screening of the Pab position for introduction resulted in a mutant that can switch its
activity with high efficiency by ultraviolet and visible light. Protein purification of the
Pab mutant showed a quality similar to that of wild-type. Detailed analysis of the KcsA
Pab mutant reveals the relationship between pH and light dependence. These results
suggest that Pab introduction may be a useful method to confer light dependence tovarious proteins.

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“Molecular Movies” international symposium 2023

Development of adenosine A2A receptor-selective photo-switchable ligands

Tomohiro Doura (Nagoya University)
Subtype-selective photo-switchable ligands are considered a kind of powerful molecular
tools for photopharmacology or optochemogenetics of G-protein coupled receptors(GPCRs). Here we report two kinds of adenosine A2A receptor (A2AR)-selective ligandswith photo-switching properties. One A2AR-selective ligand is termed photoAd(vio)which is isomerized to the cis-isomer by ultraviolet (UV) light irradiation. Because thecis-isomer of photoAd(vio) activates A2AR selectively, photoAd(vio) allows to control
A2AR in living cells with high spatiotemporal resolution. The other A2AR-selective
ligand is named photoAd(blue). The cis-isomer of photoAd(blue) is isomerized to thetrans-isomer which activates A2AR selectively by blue light irradiation. The thermallystable trans-isomer of photoAd(blue) is expected to be exploited in the structural analysis
of A2AR-selective photo-switchable ligands.

Crystal structure of photoresponsive ligand bound adenosine A2A receptor

Tsuyoshi Araya (Kyoto University)
The rational synthetic expansion of photoresponsive ligands is important for thedevelopment of G protein-coupled receptors (GPCRs) photopharmacology. In this study,
wesynthesized

the

novel

photoresponsive

andreceptor

selective

ligandphotoNECA(blue) for adenosine A2A receptor and determined the crystal structure of
the ligand-bound receptor complex. This complex structure could explain the binding
mode and the photoresponsive mechanism of photoNECA(blue). And we mentioned thepossibility

that

the

photoresponsive

ligandcould

beapplied

not

only

to

photopharmacology but also to the dynamic structural analysis of GPCRs from this study.

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“Molecular Movies” international symposium 2023

<A01_Structual biology>
Fri. 1st December / 9:00 -10:40am JST / Room 403

Real-Time Structural Changes during the Light-induced Water Oxidation of
Photosystem II Caught by Time-Resolved Crystallography

Michihiro Suga (Okayama University)
Photosystem II catalyzes water oxidation by capturing sunlight energy at the uniquepair of Chlorophylls called P680 within a picosecond time scale that initiates a serial
reduction/oxidation reaction between two plastoquinone, QA, and QB, a radical active
tyrosine residue Yz, a catalytic manganese cluster, and substrate water molecules. This
reaction is the so-called Si-state cycle catalyzed by the Mn4CaO5 cluster, which
incorporates an extra oxygen O6 at the S3 state to form a possible di-oxygen. The
structural changes of the metal cluster and its environment have been examined at themillisecond time range.

An Attempt at Elucidating the Reaction Mechanism of Cytochrome c
Oxidase Using Simultaneous Measurement of XES and XRD

Atsuhiro Shimada (Gifu University)
Cytochrome c oxidase (CcO) has four essential redox metal sites crucial for its protonpumping and O2 reduction reaction. It has been suggested that unidirectional protonpumping across a membrane utilizes the structural changes of protein moieties driven
by the redox changes of metal sites during the O2 reduction reaction. To elucidate thereaction mechanism of CcO, it is necessary to comprehend the correlation between theelectron states of metal sites and the protein structure. For the sake of this objective, I
endeavored to simultaneously acquire XRD data (atomic coordinates) and XES data
(electron states of metal sites) using XFEL.

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“Molecular Movies” international symposium 2023

Photoresponse mechanism of a bifunctional cryptochrome

Junpei Yamamoto (Osaka University)
Photolyase/cryptochrome superfamily (PCSf) proteins are flavoproteins bearing flavinadenine dinucleotide (FAD) as a catalytic/photoreceptive center and involved in various
biological functions such as DNA repair, signal transduction, entrainment of circadianrhythm, and so on. An animal-like cryptochrome from Chlamydomonas reinhardtii(CraCRY) is a unique protein responsible for both photolyase and cryptochrome
functions. In this study, we intended to unravel dynamic behaviors of light-dependent
DNA repair and FAD photoreduction via time-resolved serial femtosecond X-ray
crystallography. Intermediate structures in the ns-ms region during the FAD
photoreduction of CraCRY revealed molecular mechanism of unfolding of the C-terminal
region.

Fast time-resolved structural analysis of light-response in photosynthetic
antenna protein Phycocyanin

Yasufumi Umena (Synchrotron Radiation Research Center Division of Synchrotron
Radiation)

The cyanobacterial antenna protein, phycocyanin (PC), captures and supplies lightenergy to the reaction center, photosystem II. PC is composed of alpha and beta subunitsof Mw 20000 with three pigment molecules, phycocyanobilin (PCB). In this study, weinvestigated the light-energy transfer inside the PC in sub-picosecond using pump-probe
serial femtosecond crystallography (pp-SFX) with XFEL. The fast transient-absorption
spectra using a PC microcrystal provided proper time-points for time-resolved pp-SFX
measurement. The pp-SFX after four picoseconds showed structural changes and laser
intensity-dependent damages in PCBs at 1.6 Å resolution. We will discuss the results
and current issues in this study.

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“Molecular Movies” international symposium 2023

Reaction mechanism of photoactivated adenylate cyclase OaPAC bydynamic structural analysis

Sam-Yong Park (Yokohama City University)
Cyclic-AMP is one of the most important second messengers, regulating many crucial
cellular events in both prokaryotes and eukaryotes, and precise spatial and temporalcontrol of cAMP levels by light shows great promise as a simple means of manipulating
and studying numerous cell pathways and processes. The photoactivated adenylate
cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminatea (OaPAC)
is a small homodimer eminently suitable for this task, requiring only a simple flavinchromophore within a BLUF domain.
The structure of OaPAC has been capturing dynamic structural changes as molecularmovies on a wide range of time scales by structural analysis using synchrotrons.

Calcium binding induced structural changes required for the photoprotein
Aequorin luminescence

Toru Nakatsu (Wakayama Medical University)
Aequorin is a Ca2+-binding photoprotein that was isolated from the luminous jellyfish
Aequorea victoria and emits light by an intramolecular reaction upon binding with Ca2+.
Ca2+ binds to three EF-hand motifs of Aequorin, showing a helix-loop-helix structure.
Aequorin consists of apoaequorin and 2-peroxycoelenterazine, when Ca2+ binds
aequorin, non-covalent bounded 2-peroxycoelenterazine decomposes into coelenteramide
and CO2 to produce blue light. In this study, time-resolved X-ray crystallography of
Aequorin using SACLA and SPring-8 will reveal in order to elucidate the order of calcium
binding to EF-hand motifs and the mechanism of structural change associated calcium
binding.

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“Molecular Movies” international symposium 2023

Anaerobic X-ray diffraction data collection using oxygen barrier film forstudy on nitric oxide reductase

Takehiko Tosha (University of Hyogo)
Bacterial nitric oxide reductase (NOR) is responsible for the elimination of cytotoxic NO.
In order to elucidate the mechanism of NO decomposition at the heme/non-heme ironbinuclear center of NOR, we designed experimental systems for anaerobic data collectionof X-ray diffraction because the NOR-catalyzed reaction initiates the reaction of the
reduced form of NOR and NO. To keep the anaerobic condition during the X-ray
diffraction experiments, we focus on an oxygen barrier film to avoid the oxygen
permeation. Ethylene vinyl alcohol copolymer-based oxygen barrier film showed apotential for anaerobic X-ray diffraction experiments.

A molecular movie of photoswitchable fluorescent protein

Eiichi Mizohata (Osaka University)
The photoswitchable green fluorescent protein, rsGamillus, presents unique properties
distinct from existing photo-switchable fluorescent proteins. For example, the isomericforms of its chromophore in on/off states are trans/cis, which is opposite to that of otherphoto-switchable fluorescent proteins. In this study, I report the successful capture of amolecular movie of the photo-isomerization reaction for off→on switching of thersGamillus chromophore. This was achieved using time-resolved serial femtosecond
crystallography (tr-SFX) with a nanosecond laser-based pump-probe technique,capturing the events from 10 nanoseconds to the microsecond timescale.

Structure and mechanism of a Diels-Alderase and a 2-oxoglutaratedependent dioxygenase

Shingo Nagano (Tottori University)
A Diels-Alderase, Fsa2, which is involved in equisetin biosynthesis, produces a decalin

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“Molecular Movies” international symposium 2023

skeleton by stereo-selective 4+2 cyclization reaction. We determined the crystal structureof equisetin-bound Fsa2 and confirmed that the equisetin binding mode was consistent
with the substrate binding mode previously predicted by a molecular dynamics
simulation.
TauD, which hydroxylates taurine, is a non-heme iron- and 2-oxoglutarate-dependent
dioxygenase. Metal-free and taurine-bound TauD microcrystals were mixed with FeSO4
solution to induce the enzyme reaction. The occupancy of the non-heme iron was timedependently increased, confirming the possibility of non-heme iron reconstruction of
crystalline TauD. In addition, the polder omit map with a delay time of 9.0 sec confirmed
the electron density extending from the carbon atom to be hydroxylated in substratetaurine toward non-heme iron, suggesting that taurine was hydroxylated in the
microcrystals.

< B01_Molecular Movie Platform Design>
Fri. 1st December / 9:00 -9:20am JST / Room 404

Development of an in-vacuum diffractometer for protein microcrystallography

Hiroaki Matsuura (RIKEN SPring-8 Center)
Microbeams available at synchrotron and FEL facilities have enabled structure
determination from tiny protein crystals. However, due to fewer unit cell copies in suchtiny crystals, diffraction intensities become significantly weaker. This weak signal
complicates the observation of scattering from sample crystals, particularly as
background scattering from the air becomes prominent. We have been developing an invacuum diffractometer designed for micro-crystallography with crystals smaller than
1µm/side. Test experiments were conducted using cytidine crystals to compare datacollection under atmospheric and vacuum conditions. Our findings indicate that datacollection under vacuum conditions improves crystallographic statistics such as <I/σI>
or R factors.

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“Molecular Movies” international symposium 2023

Low background graphene sample holder for XFEL-based crystallography
and imaging

Akihiro Suzuki (Hokkaido University)
In the 2010s, X-ray free-electron laser (XFEL) facilities were constructed around theglobe to pioneer new X-ray science by taking advantage of its highly brilliant and shortradiation pulses. Single particle imaging of a protein that is difficult to crystallize wasone of the driving forces behind the XFEL constructions. However, even after more thanten years in operation, detecting diffraction signals from a single particle is still a severe
challenge. To improve the signal-to-noise ratio of diffraction signals, we have beendeveloping a low-noise sample holder using graphene instead of liquid jets for sample
delivery. In this presentation, we will present the current status of sample holder
development and the latest result of the XFEL experiment.

<C01_Spectroscopy >Fri. 1st December / 9:20 -10:00am JST / Room 404

Stepwise calcium binding in photoluminescence protein; aequorin, revealedby the time-resolved spectroscopy

Tetsunari Kimura (Kobe University)
The photoluminescence protein; aequorin (AQ) has three Ca2+ binding sites with loop
structure and the binding induces the transient emission of blue light. The stepwise
Ca2+ binding has been proposed based on the time-resolved serial-femtosecond x-ray
crystallography and serial soaking experiments. The comprehensive spectroscopic
measurement is critical to evaluate the proposed mechanism. In this study, kinetic
measurement of luminescence intensity along the AQ-Ca2+ binding and Ca2+ affinities
were investigated by stopped-flow and isothermal titration calorimetry, respectively.
Kinetic analysis clarified that the first Ca2+ binding induced the conformational change,
which was followed by the second Ca2+ binding with higher affinity, indicating that theconformational change in the loop structure was necessary to accommodate Ca2+ ion, as
proposed in structural biological experiments.

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“Molecular Movies” international symposium 2023

Structural features of Lumi intermediate on a primate blue-sensitive visual
pigment revealed by FTIR

Yosuke Mizuno (Nagoya Institute of Technology)
Cone pigments are photoreceptors responsible for color vision. Lumi, an earlyphotointermediate, of a primate blue pigment (MB) is deprotonated at the retinal Schiffbase, which is an unusual reaction not seen in other visual pigments. Here, we report
the structural features of Lumi of MB measured by FTIR spectroscopy. Site-directedmutagenesis study reveals that the Schiff base proton transfers to the E113, a counterion,
coupled with local perturbation of α-helices. Hydrogen bond strengths of T118 and C211
together with internal water molecules change upon the formation of Lumi, and thus
these hydrogen bond alterations might be key elements enabling early proton transfer
reaction.

A Unique Molecular Property of a Sodium-Pumping Rhodopsin in
Krokinobacter eikastus

Yuji Furutani (Nagoya Institute of Technology)
Microbial rhodopsins exhibit various functions such as light-driven ion pumps, lightsensors, light-gated channels, and light-activated enzymes. In 2013, a sodium-pumping
rhodopsin was found from Krokinobacter eikastus (KR2). Interestingly, a substratesodium ion is not bound near the protonated Schiff base region, which is transiently
incorporated into the protein moiety upon formation of the intermediate states. Thus,
the molecular mechanism has been extensively studied by using various techniques. In
this short talk, I will present our recent finding regarding importance of the covalentbond connecting the retinal chromophore in KR2 (S. Ochiai et al. Biochemistry 2023).

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“Molecular Movies” international symposium 2023

Excited state dynamics in a single protein crystal by using femtosecond
transient absorption microscopy

Tetsuro Katayama (Tokushima University)
Energy transfer reactions are important in photosynthetic systems as natural systems
and solar cells as artificial systems. In this study, femtosecond transient absorption
microscopy was utilized to elucidate the energy transfer reaction behavior between dyes
in a single crystal. In this presentation, we will discuss the details of the excited-state
relaxation process between dyes from the viewpoint of polarization dependence.

< C01_Computational Chemistry>Fri. 1st December / 9:00 -10:40am JST / Room 405

Development of non-adiabatic QM/MM molecular dynamics method

Kiyoshi Yagi (RIKEN Cluster for Pioneering Research)
We have developed a non-adiabatic molecular dynamics (MD) method based on QM/MM
calculations. QM/MM is a multiscale method that treats the reaction center by quantum
chemical (QM) method and the biological environment as a classical force field (MM).
The QM calculation of the electronic excited state, which is the computational bottleneck,is highly parallelized to achieve high performance. In addition, QM/MM is combined witha surface hopping algorithm proposed by Zhu and co-workers, which takes into accountnon-adiabatic transitions between electronic states. The method is applied tophotochemical processes of azobenzene in solution and bacteriorhodopsin.

Overview of our research collaborations performed in the research area of“molecular movies”

Mitsuo Shoji (University of Tsukuba)
We have performed collaborative researches under the research area of “molecular

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“Molecular Movies” international symposium 2023

movies”. The target molecules and collaborators are (1) C-phycocyanin with Y. Umena(A01), (2) copper amine oxidase with T. Murakawa (A01), (3) heliorhodpsin with K.
Katayama and H. Kandori (A01), (4) lysozyme with B. Maity and T. Ueno (B01), (5) 2oxoglutarate-dependent dioxygenase with S. Nagano (A01) and (6) P450 sesamin with E.
Mizohata (A01). We overview all of these research progresses, achievements andremaining issues.

How the mGLU1 receptor dimer be activated: a molecular dynamics study

Duy Phuoc Tran (Tokyo Institute of Technology)
We present in this study our recent progress in understanding the activation mechanismof the mGLU1 receptor. In collaboration with Kiyonaka group, we have figured out themechanism of the activation of the mutations and ligands. In addition, we reconstructed
the full-length structure of the mGLU1 dimer with Gi1 protein by molecular docking andsimulation. The data show the differences in interaction interfaces between mGLU1 andthe other mGLU receptors in sub-family.

Complementing molecular movie by analyzing free energy landscape,pathways, and flux of conformational change

Akio Kitao (Tokyo Institute of Technology)
We conducted molecular simulation of biomolecular systems to complement molecularmovie experimentally determined. Using parallel cascade molecular dynamics (PaCSMD) simulation and other computational techniques, we investigated conformationalchange of proteins by analyzing free energy landscape, transition pathways and flux. We
report interfacial activation of Candida antarctica Lipase B and molecular mechanismsof structure support and smooth rotation in the bushing/rod complex of bacterial flagella.

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“Molecular Movies” international symposium 2023

Dynamical Analysis for Simulations of Orexin 2 Receptor Using Relaxation
Mode Analysis

Ayori Mitsutake (Meiji University)
We have introduced and developed algorithms for protein simulation. For dynamics andstability, we introduced relaxation mode analysis and 3D-RISM theory, respectively. 3DRISM theory estimates the solvation free energy. We use molecular dynamics
simulations and these analyzes to study the inactive and active conformations of theorexin-2 receptor. From simulations of constitutively active mutants, we identified a good
indicator for distinguishing between inactive and active conformations of class A GPCRs,
the distance between Ile48.CA (3.46) and Tyr364.CA (7.53). From simulations of active
and inactive structures in the presence and absence of ligands, we investigatedcharacteristic amino acids that are thought to be important for activation of orexin-2
receptors.

Theoretical study on molecular mechanism of an activation process of
aequorin bioluminescence

Shigehiko Hayashi (Kyoto University)
Aequorin is a bioluminescent protein which binds coelenterazine as a light emitting
molecule. A chemiluminescence process of coelenterazine with a molecular oxygen in theprotein binding pocket is triggered by binding of calcium ions at EF-hands of the protein
distant from the binding pocket. We theoretically investigate molecular mechanism of
the chemiluminescence process in the protein by means of hybrid QM/MM molecularsimulations. We found significant conformational changes of the binding pocket upon thebinding of calcium ions and a proton exchange through hydrogen-bond network formedbytheconformationalchangeswhichchemiluminescence.

25

producesastableintermediatefor“Molecular Movies” international symposium 2023

Unification of molecular movies and quantum molecular dynamics

Junichi Ono (Waseda University)
Proton transfers associated with structural changes play a vital role in function. Recently,
molecular movies of structural changes in biomolecules have been successfully captured
using time-resolved SFX at XFEL. However, direct observation of proton transfers has
not been accomplished because of the limitation of spatio-temporal resolution. Here,large-scale quantum molecular dynamics (QMD) simulations were performed on thebasis of the molecular movies with focusing on the chemical reactions in rhodopsins,
where all the atoms were treated quantum-mechanically. In this presentation, we
discuss our results demonstrating that our QMD simulations compensate for themolecular movies to reveal the microscopic mechanisms of chemical reactions.

Resi-DEM: A Comprehensive Tool for Automated Density Extraction,
Identification, and Analysis of Difference Densities in Isomorphous Maps,
with Occupancy-Estimation for Conformer Fitting

Sriram Srinivasa Raghavan (RIKEN Center for Computational Science)
We will introduce Resi-DEM, an new tool designed to automate the identification ofdifference densities in isomorphous difference density maps, further associating themwith relevant residues through a clustering approach. Utilizing the clustered difference
density features, we estimated the occupancy of the triggered state population via thestructure factor extrapolation technique. This clustering technique was employed toorganize difference densities, enabling us to represent them as networks, facilitating the
tracking of density changes over various time periods. Moreover, we employed molecular
dynamics trajectory sampling to determine the most fitting conformation corresponding
to the triggered data set. The results were validated using metrics such as the Real Space
Correlation Coefficient (RSCC) and Real Space Difference Density Z score (RSZD).

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“Molecular Movies” international symposium 2023

Analysis on the dynamics of bacteriorhodopsin using MD simulation

Keiko Shinoda (The Institute of Statistical Mathematics)
In the “molecular movies”, we have developed force fields for lipid molecules in thearchaeal membrane and constructed an archaeal model membrane. Using this, we havecarried out molecular dynamics (MD) simulations of bacteriorhodopsin. In thispresentation, in addition to the previously reported results on the resting state and at
16ns after excitation of bacteriorhodopsin, I will report on the results at 760 ns and 2µs
after excitation and present a comprehensive analysis on the dynamics ofbacteriorhodopsin.

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