Cheiron School, 8 November 2009, SPring-8

Protein Crystallography:
Synchrotron X-rays
i questt off new horizons
in
h i
in
i
the protein universe
7 GeV & 4 GeV KEK-B ringg (⇒( KEK-X )ERL
cERL

6.5GeV PF-AR
2.5 GeV PF

Soichi Wakatsuki
Photon Factory
IMSS, KEKRole of Structural Biology
TranscriptionpGenomeDNA→mRNA→

Proteins
Translation

Molecular machines（ribosomes,
enzymes）
oo networke woInformation

Proteintransport

Atomic resolution analysisy○Protein structure and dynamics○Interactions between molecular machines
Signaltransduction

Biology(Basic research)

Medicine, drug design
Medicine(industrial applications)

Ribosome

The Worldwide Protein Data Bank (wwPDB)

http://www.wwpdb.org/index.html
50,000 entries and growing!

David S. Goodsell, Scripps InstituteHow large
g is the pprotein universe?
• How many genes?
– MetaM t genomes --- J.
J C
Craig
i VenterV t Institute's
I tit t 'Global Ocean Sampling Expedition– Human microbiomes (Gill, et.al. David Relman,
Stanford, Science 2006)– Emerging infectious diseases• Splicing variants – exon: cut and paste• Non-coding RNA: largely uncharted• Protein-protein, protein-carbohydrate,
protein-lipid
proteinlipid, protein-nucleic
protein nucleic acid interactions• Posttranslational modifications

Panoramic view of aeukaryotic cell
http://www.scripps.edu/mb/goodsell/
Movements

COLORS: proteins in blue, ribosomes inmagenta, DNA and RNA in red and orange,li id iin yellow,
lipidsll
andd carbohydrates
b h d t iin green.
David S. Goodsell, Scripps InstituteProtein-protein interaction network

Taken from Rua et al., Nature 2005

Protein life cycle and posttranslational
modification
mRNA ER Protein transport<-> Golgi

Apparatus

DNA
Mature proteins

Immature proteins

Posttranslational
P
tt
l ti
l modification:
difi tiglycosylation
NucleusF
Functionaltil

To destinations

Refolding by chaperons
Degradationeg ada o by pproteasomeso easo es
Degradation by lysosome/autophagy

Programmed cell
death (apoptosis)

What are posttranslational
modifications of proteins?
• Modification
M difi ti off proteinst i after
ft biosynthesisbith i• Not directly
y coded in the DNA sequencesq• Add functionality, modify surface properties,
transport signals– Glycosylation– Ubiquitylation (SUMO,(SUMO NEDD etc.)
etc )– Lipidation– PhosphorylationPhh l i– Acetylation– And moreGlycosylation

Ribosomes
ER

Cis-Golgi

GlcNAclMannoseGlucose
Phosphate
FucoseGalactose
Sialic acid

Medial-Golgi

Trans-Golgi

Model for intracellular transport of N-linked glycoproteins

Model for binding between VIP36 and high-mannose typeglycoprotein, salivary αα-amylase
amylaseTadashi Satoh, Nathan Cowieson, et al., J. Biol. Chem. (2007) 282, 28246

Structural studies towards new Influenza virus inhibitors
Hemagglutinin
Neuraminidase

Entry

Release of
new virions
Host cell surface

Glycoprotein withsialic acidStructure Based Drug Design：more efficient and lower cost for development of
Anti influenza drug with little or no side effects (collab. with Italy, Australia, Canada)
Influenza neuraminidase

TamifluT
ifl
derivedLead compounds

Zanamivir。
Inhibitor design

Clinical tests
Selection

Active site

Drugs with noside effects:
Interaction with
only influenza
neuraminidase but
NOT with humansialidases

Structure Determination using SR
Tamiflu
Difference

Yellow: human sialidase Neu2
Gray: Influenza neuraminidase

Human sialidase
Neu2 structure

Side effects:
Humansialidases?
Neu1, Neu2,
Neu3, Neu4

Flow of protein structure analysis（２）Crystal evaluation/harvesting system

CrystalliCt llizationrobot:
nucleation（１）Large scale protein
expression and（３）High brilliance microfocus MAD beam linepypurification system
Minipole Undulator

PF-2.5 GeV SR Ringg

Automated
crystal exchange
robot andalignment system
inside the hutch（４）Automated（４）A tt d proteint i crystal
t l structuret tanalysis suite2D area detectorATG AAGGuanine nucleotide exchange factor Sec12p
1 atgaagttcg tgacagctag ttataacgtc gggtatcctg cgtacggtgc aaaatttttg
M K
61 aataacgacatcattacttgt
tt tt t ggcaggcggt
t ggaggagaag gaaacaatggtcataccaaact

MKFVTASYNVGYPAYGAKFLNNDTLLVAGGGGEGNNGIPNKLTV

121 aagctgacgg tcttgcgcgt ggatcctacc aaagatactg agaaggaaca gtttcatata

LRVDPTKDTEKEQFHILSEFALEDNDDSPTAIDASKGIILVGCNENSTKITQGKGNKH

181 ttgagcgagt ttgcattgga agacaacgac gactctccta ctgcaattga cgcttccaag

LRKFKYDKVNDQLEFLTSVDFDASTNADDYTKLVYISREGTVAAIASSKVPAIMRIID

241 ggtatcattt tggttggctg caatgaaaat agcactaaga ttacccaagg taaaggtaat

PSDLTEKFEIETRGEVKDLHFSTDGKVVAYITGSSLEVISTVTGSCIARKTDFDKNWS

301 aagcacttga gaaaatttaa atacgataaa gtgaatgatc aattggagtt cctcactagt

LSKINFIADDTVLIAASLKKGKGIVLTKISIKSGNTSVLRSKQVTNRFKGITSMDVDM

361 gtagactttg acgcatctac aaatgcggat gactacacga agctggttta tatttcacga

KGELAVLASNDNSIALVKLKDLSMSKIFKQAHSFAITEVTISPDSTYVASVSAANTIH

421 gaaggtaccg ttgcagctat cgcatcatct aaagtacctg ctataatgag aatcattgac

IIKLPLNYANYTSMKQKISKFFTNFILIVLLSYILQFSYKHNLHSMLFNYAKDNFLTK

481 ccgagcgact tgacagagaa gtttgagatc gagactaggg gtgaagtaaa ggatttacac

RDTISSPYVVDEDLHQTTLFGNHGTKTSVPSVDSIKVHGVHETSSVNGTEVLCTESNI

541 ttttccactg
g atggtaaggt
gg
gg tgttgcttat
g g
atcaccggtt
gg
ctagcttggag
gg agtgatttca
g g

INTGGAEFEITNATFREIDDA

601 acagtgactg gaagttgcat tgctaggaaa acagattttg ataagaattg gagtttatct
661 aaaataaact tcatagccga tgacacagta ttgatagcag cctctttaaa aaaagggaaa

No of amino acid residues：471

721 ggtattgtgc tgaccaaaat aagcatcaaa tcaggaaaca cttccgtatt aagatccaaa
781 caagtgacaa acagattcaa agggattact tctatggatg tcgacatgaa gggtgaattg
841 gcggtactgg caagtaatga caattccata gctcttgtga aactaaaaga cctgtcaatg
901 tctaaaatat tcaaacaagc tcatagtttt gccattacag aggtcactat ctctccggac
961 tctacatatg tggcgagtgt ttcggcagcc aacactatcc acataataaa attaccgctt
1021 aactacgcca actacacctc aatgaaacaa aaaatctcta aatttttcac caacttcatc
1081 cttattgtgc tgctttctta cattttacag ttctcctata agcacaattt gcattccatg
1141 cttttcaatt acgcgaagga caattttcta acgaaaagag acaccatctc ttcgccctac
1201 gtagttgatg aagacttaca tcaaacaact ttgtttggca accacggtac aaaaacatct1261 gtacctagcg tagattccat aaaagtgcat ggcgtgcatg agacgagttc tgtgaatgga
1321 actgaagtct tatgtactga aagtaacatt attaatactg gaggggcaga gtttgagatc
1381 accaacgcaa cttttcgaga aatagatgat gcttga

No. of bases：１４１６（1416‐３）/３＝４７１

Model structure of Sec12

Phase Determination using Multiple Anomalous Dispersion
Methionine
SulfurSelenium (SeMet)

M K
MKFVTASYNVGYPAYGAKFLNNDTLLVAGGGGEGNNGIPNKLTVLRVDPTKDTEKEQFHILSEFALEDNDDSPTAIDASKGIILVGCNENSTKITQGKGNKHLRKFKYDKVNDQLEFLTSVDFDASTNADDYTKLVYISREGTVAAIASSKVPAIMRIIDPSDLTEKFEIETRGEVKDLHFSTDGKVVAYITGSSLEVISTVTGSCIARKTDFDKNWSLSKINFIADDTVLIAASLKKGKGIVLTKISIKSGNTSVLRSKQVTNRFKGITSMDVDMKGELAVLASNDNSIALVKLKDLSMSKIFKQAHSFAITEVTISPDSTYVASVSAANTIHIIKLPLNYANYTSMKQKISKFFTNFILIVLLSYILQFSYKHNLHSMLFNYAKDNFLTK
RDTISSPYVVDEDLHQTTLFGNHGTKTSVPSVDSIKVHGVHETSSVNGTEVLCTESNIINTGGAEFEITNATFREIDDA

No. of amino acid residues: 471
including 6 methionines
A ribbon representation of a protein with70 selenium atoms superimposed incolour Equivalent selenium atoms fromcolour.
molecule to molecule are coloured thesame.

Protein crystallization by vapor
diffusion method
Vapor equilibrium

Precipitant solution

Protein solution drop (containing water, precipitant and protein molecules)
Precipitant molecule
Water molecule
Protein crystal

Drawing by Prof. Yoshiki Higuchi

~1012 proteins in a typical (good size) crystal

Crystal of
Human GGA1
VHS domain

bar＝0.1 mm
Crystallization method: hanging drop vapor diffusion
Protein conc.:
13 mg / ml
Precipitant:p
17 % ((w/v)) PEG3350, 0.2 M KH2PO4
Buffer:0.1 M Tris-HCl (pH 7.5)
Temperature:

20 ºC

Packing
g of pproteins in a crystal
y30~70 % volume of protein crystals is solvent!

Cocrystal

Peptide-tagged

Why synchrotron?
• X-rayX
i tintensityit• Parallel beam
• Micro beam

λ tunabilityy⇒ measurements
t off
weak reflections⇒ high-throughput
measurements⇒ large unit cells⇒ micro crystals⇒ use of best part ofcrystals⇒ pphase determination

Intensity of a reflection
4
Vx
e
2
3
E (hkl ) = 2 4 I 0 λ LPA 2 F (h )m cωV0F(h): structure factor
ω: speedpof crystal
yrotation
I0: Intensity of incoming X-ray
λ: wavelengthg
P: polarization factor
L: Lorentz correction
A：absorption correction
Vx: volume of the crystal
yin the beam
V0: unit cell volume

Methods to Determine Phases
Heavy atom ＭＡＤ(Multiple Anomalous Dispersion)

Light atom ＳＡＤ(Single Anomalous Dispersion)

Introduction of
anomalous
scatterers

Wild type
crystals

・Seleno-methionine
Seleno methionine (SeMet)
・Heavy atom soaking

λ１
λ2
λ3

X-raysλX-raysMulti wavelength anomalous dispersion

Anomalous signal from light atoms (eg: S)

・Good
Good method as long as heavy atom derivatives
are available
・Multiple data sets need to be collected quickly
・Possible to use for extremely large complexes・Noo needeed foro ppreparing
epa g heavy
ea y ato
atoms:
s highlyg yapplicable for very difficult cases for which heavy
atom derivatives cannot be prepared・Light elements need low X-ray energy for higher
anomalous signal
g・Weak anomalous signal necessitates extremely
highly accurate data, thus high redundancy

Phase determination using MAD
Bijvoet pairs(M lti l A(Multiple
AnomalouslDi
Dispersion)i )Bijvoet Differences

MAD experimentalil maps:Anomalous difference Patterson

E2
E1

Se K edge

ΔFE32 (at the peak)
Dispersive Patterson
FE42 - FE22 (remote – inflection point)

E3
E4

Oscillation method:(PRESENT)(eg. 90 X 1 deg oscaillation)Datatransfer
Insertion device

SR Ring

2D area detector

1 degree oscillation

FPH(SeMet)-FP(native) difference
Patterson Map

FPH(SeMet)-FP(native) anomalous
difference Patterson Map

FE3(+)-FE3(-) anomalous difference
Patterson Map

FE4(remote1)-FE2(edge) dispersive
difference Patterson Map

FPH(+) = FP + FH(+) = FP + FH(Re) + FH(Im)FH(+)

FPH(-) = FP + FH(-) = FP + FH(Re) -FH(Im)

FH(-)
FP

Measurements:

FPH(+)

FPH(-)|FP |at remote energy, E4|FPH(+) |, |FPH(-) |at the peak of f”, E3

From the heavyy atom ppositions:
FH(+) & FH(-)

From intensity measurements
|FP|

Im

|FPH(-) ||FPH(+) |ϕP (?)Re

From Se positions-FH(+)-FH(-)|FP|

Im|FPH(+) |-FH(+)
-F
FH(-)
()

ϕP
Re
|FP|

|FPH(-) |Met94

Met84

Ribbon diagram of trimer of GGA1 GAT domain
Triangle is threefold axis.

Large complexes requires high brilliance (high intensity &parallel beam) and large-area detectors.
Robot IP changer
Kappa diffractometer
MAR CCD 133 mm

X ray Beam
X-ray

Flat IP detector wall

Inside ID14 EH3 of the ESRF, Grenoble

Bluetongue virus structure analysis by D.Stuart, Univ of Oxford

ID2PX ESRFID2PX,

Diffraction data on large IP
detector on EH3/ID14, ESRF

Shown the possibility of 2000 unit celldimensions
Even larger with ultra low emittance?

Blue Tongue Virus
Core Particle 1
P21212755 X 796 X 825 Å3
ID14/EH3570 Å(20 ピクセル)
ピクセ

Crystal todetector
distance

1250
mm

Wavelength

0.918Å

Oscillation

0.1 deg

Exposure time

100 sec

Pixel size

100 mm

B
Beam
size
i

100 mm

FWHM

181 mmhttp://www.kek.jp/ja/news/topics/2009/NobelYonath.html

Nobel Award in Chemistry
Chemistry, 2009

• Ribosome structures• Prof.
Prof Ada Yonath was a user of the Photon Factoryfor 10 years from 1987
• Her plenary lectures– Biology and Synchrotron Radiation/Medical Applicationsin SynchrotronyRadiation in Melbourne between Februaryy
15-19, 2010
– Symposium of Target Protein Resarch Program March in
T kTokyo,
on 5th, 2010
– Photon Factory Symposium in Tsukuba on March 9, 2010

Prof. Ada Yonath( 2nd from right) & Prof Noriyoshi Sakabe (3rd from left)
In ICCBM conference in Beijing, June 2004

http://www.kek.jp/ja/news/topics/2009/NobelYonath.html

Prof. Ada Yonath （left）. At a BSR92（4th international conference on Synchrotron
and Biology） with Prof. Wayne Hendrickson of Columbia University, USA （center）、
仏Dr. Joseph Zaccai of Laue-Langevin Insittute, Grenoble, France（right）.

http://www.kek.jp/ja/news/topics/2009/NobelYonath.html

Cryo cooling apparatus developed by Prof. Ada Yonath （courtesy of Prof. Sakabe）

Number of unit cells in crystalsyUnit cell dimension
Crystal size
( i(micro
cube)

30Å

40Å

50Å

100Å

200Å

300Å

500Å

800Å

300

1.0E+15

4.0E+142.0E+143.0E+13

3.0E+12

1.0E+122.0E+115.0E+10200

3.0E+141.0E+14

6.0E+138.0E+12

1.0E+123.0E+11

6.0E+102.0E+10100

4.0E+132.0E+138.0E+12

1.0E+12

1.0E+11

4.0E+108.0E+09

2.0E+0950

5.0E+122.0E+12

1.0E+12

1.0E+112.0E+105.0E+09

1.0E+09

2.0E+0830

1 0E+12
1.0E+12

4 0E+11
4.0E+11

2 0E+112.0E+11

3 0E+103.0E+10

3 0E+09
3.0E+09

1 0E+09
1.0E+09

2 0E+082.0E+08

5 0E+07
5.0E+0720

3.0E+11

1.0E+11

6.0E+108.0E+09

1.0E+09

3.0E+08

6.0E+072.0E+0710

4.0E+102.0E+108.0E+09

1.0E+09

1.0E+08

4.0E+078.0E+06

2.0E+06

5

5 0E+09
5.0E+09

2 0E+09
2.0E+09

1 0E+09
1.0E+09

1 0E+08
1.0E+08

2 0E+072.0E+07

5 0E+06
5.0E+06

1 0E+06
1.0E+06

2 0E+05
2.0E+05

42.0E+09

1.0E+09

5.0E+08

6.0E+078.0E+06

2.0E+06

5.0E+05

1.0E+053

1.0E+09

4.0E+082.0E+08

3.0E+073.0E+06

1.0E+06

2.0E+05

5.0E+042

3.0E+08

1.0E+08

6.0E+078.0E+06

1.0E+06

3.0E+05

6.0E+04

2.0E+041

4.0E+072.0E+078.0E+06

1.0E+06

1.0E+05

4.0E+048.0E+03

2.0E+03

Numbers of “cubic shape” unit cells in cubic shape crystals. Coloredaccording to the number of unit cells contains in crystals: more than 108
copies in green, 107 pink, & 106 copies yellow. （by Masaki Yamamoto,
Harima RIKEN/SPring-8）

Extremely large complex, Vault(Tsukihara Group, Science 2009)• Structure determined at 3.5 Å reslution with 39-fold symmetry• MVP(major
MVP( j vault
lt protein)t i ) aloneliis 99kD
99kDa x 78 copies
i =7
7.7
7
MDa
• SPring-8
SPring 8 BL44XU Osaka Univ
Univ. Beam Line

Vault crystal （Tsukihara et al.,Science 2009, SPring-8 BL44XU）

Photographs of vault crystal. The crystal was about 0.70 mm x 0.15mm x 0.03 mm, and belongs to the space group C2, with celldimensions of a = 702.2 Å, b = 383.8 Å, c = 598.5 Å, and=124.7°.

10,000 x 4000 x 500 = 2 x 1010 unit cells

Micro crystals of cypovirus polyhedra• Structure
St t
att 2 Å resolution
l ti(Peter Metcalf group,
Nature 2007, datacollected at Swiss Light
Source, presented in
AsCA 2009,2009 Beijing)• Very robust crystals：
dissolve only above pH10
• a=b=c= 103 Å, １ micron
cube crystal ⇒ contains
106 unit cells ⇒ 100
repeats along each axis

Laue function of micro crystals
Limit 10 x 10 x 10 = 1000 ?

Laue function

For Na = 10

Roadmap of protein crystallography beamlines in Japan
Examples A: membrane proteins, B: enzymes without heavy atom lables& C: virus crystals

SPring-8

PF

1μm

BL32XU

BL-1A

A
BL41XU（Osaka Univ）

BL38B1
BL26B1/2

C

AR-NE3A
BL-5A
AR-NW12A

Size of ccrystalBL44XU

BL-17A

BL-6A
B（RIKEN）

SAGA
BL-7> 200μmKEK-Photon Factory (Tsukuba)
Structural Biology Research Center （since 2003）
AR NW12

Wet lab
6.5 GeV AR

PF BL5

2.5 GeV
PF-Ring

Crystallization robot
200,000 cond./day

AR NE3 Astellas

Vesicle transport andposttranslational modification
PF BL1PF BL17

5 large grants （01～09：Total
US$ ６９M、US$４０M for KEK）

Collaboration with domestic andoverseas groups from 8 countries

Low energy SAD,
microfocus

SSRL-type robot installed on MAD Beamline BL-5
20 datasets/day ⇒ 100s datasets/day
4-axis Robot
Monitoring Camera
Force Sensor
Cryo Tong

SampleGoniometer

LN2 Dewar

CCD Detector

Double Tongg

Cryo Nozzle

Cassettes

Fancy Box

Compatiblepwith SSRL（USA）, SRRC（Taiwan） &AS (Australia), CLS (Canada)
Compatibility with SPring-8 and European
standards being worked

SAM SystemyFamilyyALS
12.3.1
CLS 08B1-1(CMCF2)

KEK-PF
BL-1A, BL-5A, BL-17A,
AR-NW12A, AR-NE3A

SSRL
BL1 5 BL7
BL1-5,
BL7-1,
1 BL9
BL9-1,
1
BL9-2, BL11-1, BL11-3

NSSRC
BL13B1, BL13C1
AS
3-BM1, 3-ID

SSRL
Automated
Mounting system

But so many different systems…
MAR-CSCBAMSPACESBC CATSBC-CATEMBL-Hamburg
EMBL
Hamburg

ACTOR

EMBL-SC3

PLS

DORIS

BruNo

CATS

SAM

Cool-Hand-LukeCompatibilitypy with other systemsy

SAM System
PF willaccommodate to
Universal Pucks

BAM(ALS)

Under
development
within the Target
ProteinResearch
Project

SPACE(SPring-8)
Universal Puck

Rigaku ACTORDIAMOND, SOLEIL, SLS, APS,…

NEW BL17A short gap undulator beam line: Example of small crystals(funded by JST Frontier Technology Development Project)
1.5Å

AnothercrystalBeam Line:
Wavelength
Beam size
si e :
Exposure time:

PF-BL17A
1.291Å
20 μm
m × 20 μmm
20 sec20x20x20μm

100μm

Provided by Dr. Tadanobu Tanaka of Showa University

Highg precisionp
one axis diffractometers with XYZ stages
g
BL

BL-6A

NW12

BL-5

BL-17

Y
Year
started
t t d

2000*

2003

2004

2006

Max deviation（μm）

10

2.2

1.0

0.37(2007)⇒ 0.1(2009)

Xtal size （μm）

100

22

10

4 (2007) ⇒ 1 (2009)06
0.6

変位量 [μmm]

0.40.36μm0.2
00.6 μm

-0.2
-0.4
-0.6
0

Ai b
Air
bearingi & magnetic
ti x-y stages
t

100

200
ω角度 [degree][d]BL-5 type diffractometer -> also installed on BL41XU & BL44XU, SPring8300

No. of Beam Time Proposals on Protein Crystallography Beam Lines at PF
Doubled in the last 7 years.
250

Accepted Proposals

Sum of Running Proposals

200

150

100

50

0

01 1st01 02 1st 02 03 1st 03 04 1st 04 05 1st 05 06 1st 06 07 1st 07 08 1st
2nd
2nd
2nd
2nd
2nd
2nd
2nd

Industrial Use and Collaborations
between KEK and Industry (~16%( 16% of beamtime)

MX BLs at Photon Factory

Ajinomoto Co., Inc
Astellas Pharma Inc.

Astellas Pharma.
Beam Line ARNE3 (to becompletedl t dbbyMarch 2009)
Company ①
Company ②
Astellas ③

Tsukuba Structural
Bi l
BiologyC
Consortiumifor Industrial
Applications（sinceApril 1, 2006）
Pharmaceutical Consortiumfor Protein Structure
Analysis (PCProt), Japan
Pharmaceutical
Manufacturers Association(JPMA)
BL32B2 at SPring8

Eizai Co. Ltd.
KYOWA HAKKOKOGYO Co., LTD.
DAIICHI SANKYO Co.
Ltd.
CHUGAI
PHARMACEUTICAL
Co., Ltd.
BANYU
PHARMACEUTICAL
Co., Ltd.
Mitsubishi Chemical
Corporation
New members

Fragment Based Lead Discovery
Fragment Evolution”
Evolution“Fragment
Low MW fragments•低分子量ﾌﾗｸﾞﾒﾝﾄ
•a1
a1
High
conc assay,
NMR, SPR,•,•NMR,•SPR•高濃度ｱｯｾｲ
X-ray
crystallography•X•線結晶構造解析
線結晶構造解析

•a3
a3

•a2
a2
•a4
a4

•a5
a5

•a11
•A
A

B
•B

Chemical Library
•a1
a1

•b1
b1

•a1
a1

•a1
a1
11

•b1
b1

•A
A

•B
B

•b2
b2

Combinatorial
synthesis•ｺﾝﾋﾞ合成

Bioassay•ﾊﾞｲｵｱｯｾｲ
a1

•b3
b3
b3

a1

b5

a1

b4

Astellas Pharma Beam
Line: PF-AR
PF AR NE3
Available from Apr. 2009

NE3 Undulator

Optics Hutch
CM DCM FM

FPExp. Hutch• Stronger than AR-NW12A by a factor of 3
• Astellas Pharma will have ppriorityy access for certain
amount of beam time during 10 years from April 2009.
• The rest of the beam time can be used for general user
operation including use by other pharmaceuticalcompanies.
• High throughput: 450 datasets in 3 days

International Collaborations
3000 registered users
Australian BL20B
500~600 papers published each year
since 1992, one of themost productive BLs at
NW10A
PF(Partial use)
⇒ Also industrial use (2009~)

BL20B
BL18B
⇒IndianBL
ANBF-Photon Factory
698 Experiments~1 500 scientist visits to Japan~1,500>> 2000 Days of beamtime
Many hundreds of publications

PF-AR (6.5 GeV, 60mA, SB)
10 (8 independent) stationsFirst steering committee, IndianEmbassy, June 25, 2009
Dr. Sood’s visit to PF,
Oct 5, 2009

PF (2.5/3 GeV, 450 mA, MB(SB))
56 (48 independent) stations

Protein 3000 Project (the MEXT) 2002~2007
Network Committee forProtein Analyses
500～600 structures,
str t res R&D,
R&D
Structure-Function,
Education
Transcription andTranslation

Corporation in
structural
genomics R&D

RIKEN
RIKEN Structural Genomics/Proteomics
Initiative (RSGI)（2500 structures、Structure.-Function、R&D）
Yokohama Genome Science Center
Harima Institute

Other
Institutions

Collaboration
Collaboration

Development and Cell
Differentiation
Protein Transport andModification

Osaka Univ.
Univ
Inst. for Protein
Research

Other ResearchCenters of RIKEN
Beam time use

C ll bCollaborationiCollaboration
Collaboration

Beam time use

Signal Transduction
SPring-8
Higherg
Order BiologicalgFunctionsBrain and Neurology

Corporation
in R&D and
facility
operation

Beam time use
Photon Factory

Beam time use
Metabolism
Collaboration

Industry

Protein 3000 Tsukuba Structural Biology Consortium (21 groups)
Protein Transport and Posttranslational Modification
Showa Univ (PX)

KEK, Photon Factory (PX, SAXS)N
Nagaoka
k T
Tech
hU
Univ
i (X
(X-ray))Nara Inst. Sci. & Tech (SAXS)

Nagoya City Univ. (NMR)

Tokyo Inst. Tech (PX)JAERI, Nara (Bioinfo)

Original goal: 70 in 5 years (2003-March 2007)⇒ changed to 150 in the second year
254 structures solved (31 March 2007)
Kagoshima University
Medical School

Osaka University
Inst. Microbial Diseases
Kyotoy
UniversityyPharmacology Department
NAIST,
Molecular Biology Lab
Kyoto University
Life Science Program

U i
University
it off TokyoT k (Komaba)(Kb )Osaka University
Medical School
Tokyo Metropolitan
Institute of Gerontology

Riken, Membrane
Traffic GroupRiken
Immunology InstituteKyoto Sangyo University
Faculty of Engineering

Target Protein Project (5 years: 2007-2011)• Yearly
Y l budget:b d t 55.5
5 billibillion yen, US$ 44
44.5M,
5M 33
33.3
3 ME
MEuro,(includes 30% overhead)• 43 teams selected on 15 June 2007
• KEK: Vesicle transport & Structural Analysis Core
Targets:

Fundamental
Biology

5 yr term

7

6

5

3 yr term

5

4

6

Protein
Production Core

Medicalimportance/relevanceFood andenvironment

Structural Analysis
Core

Functional
Control Core
(Chem. Library)

Informatics Core

5 yr

1

1

1

1

3 yr

3

2

0

1

Photon Factory

MEXT: Target Protein Project(2007 2011)(2007-2011)

SPring-8

Joint Proposal by SPring-8 and PF:
Two Complementary New Beam LinesPF② Microfocus
Beam Line
Low to medium
energy (Invacuum
short gap
undulator)
1.
2.
3.

① Microfocus
Beam Line
Medium to highenergyTo be completedp
byy end of March 2009
Compatibility: SPring-8 and PF robots
Remote access and control

In collaboration with Hokkaido Univ, Kyoto
U i andd Osaka
Univ,
O k Univ.
U i

SPring-8

In-vacuum Short Gap Undulator
InMicrofocus Beam Lines: softer X
X--rays
NEW BL1A

4
P

NEW BL1A

3.5

S
Cl

3
f"(Electro
ons)

K

2.5

Ca

BL17A

2

Se

BL17A

1.5
1
0 5
0.5
0
4

5

6

7

8
9
10 11
Energy (keV)

12

13

14

Towards lower background:
Promising techniques developed by other groups.
Loopless crystal mounting method

Kitago, Y
KitagoY., Watanabe
Watanabe, N
N. and Tanaka
Tanaka, II., Acta
Cryst., D61, 1013-1021 (2005).

http://castor.sci.hokudai.ac.jp/watanabe/Xtal
Mount/

Towards lower background:
Promisingg techniques developed byy other ggroups.
OR CUT AWAY the rest with a 193 nm UV laser

Courtesy of Nikon Ltd
Ltd. & Sosho Ltd
Ltd.
H. Adachi et al., Japanese Journal of Applied Physics Vol. 44, No. 2, 2005,
pp. L54-L56 Protein

Long Wavelength MX BL at Diamond:I23 (2~4 A)

Courtesy of Armin Wagner, Talk on Friday 11:45

PF-AR NW14A: ERATO Project (~2009)
Dynamics studies with 100 ps time resolution for innovation in materials and biologicalsciencesTokyo Institute of Technology, ERATO (JST) S. Koshihara、KEK・PF Shin-ichi Adachi
Laser
induced spincrossoverreaction

Laser driven shockwaves through CdS
single crystal

Solutionphotoreaction
Dynamics of ligand migration in protein crystal

Systems Structural Biology
Posttranslational modification and transport
Plasma Membrane

GGA

Secretory Vesicles

Proteinglycosylation

Clathrin
CoatedV i lVesicleEndocytic
pathway
Early Endosome

TGN

Golgi apparatus

Late Endosome

Endoplasmic
ReticulumL
Lysosome

Melanosome
Nucleus
Autolysosome

Autophagic pathway

SmallS
ll GTPases,
GTP
motor
t proteinst iinvolved in vesicle transport:
R b and
Rab
dA
Arf,
f ki
kinesin,i anddclathrinSmall GTPases, ARF and Rab, in vesicular trafficking
T. Shiba, et al., Nature Structural Biology 10 386
386-393,393, 2003

T. Shiba et al. PNAS 103, 15416, 2006

Taken from I. Jordens, et al., J.Neefjes, Traffic 2005; 6: 1070–1077

N. Hirokawa, Univof Tokyo Med
SchoolFrom N. Hirokawa, J. of Neuroscience, 2006

Transport in nuerons

DoubleheadedkinesinOgawa, et al., Cell, 2004

~0.5 μm

1~2 μm/sec
8 nm step

KIF1A: single headed motor, Nitta, Hirokawa et al., Science
2004, 305, 678 – 683, Data collected on PF-BL6A & 19ID APS

Higher order protein complexes localized on organelle
membranes
EM
Confocul microscopy (live cell imaging)in vitro reconstruction system
Synchrotron X-ray diffraction imaging
AP, & GGA
Clathrin

BAR domain

ESCRT-III
Hi h resolutionHighl ti proteint i structurest tMultiple Arf/Rab interactions with their effectors

Organelle morphology
Organelle structure and function

Golgin: multi effector protein for Arf/Rab GTPases
Golgin proteins (GCC185）
Very long coiled-coil structure
Scaffold for maintaining Golgi stacks

Rab small GTPases (> 60) are localized on specificorganelles
GCC185
GCC185

Golgi Stack

GCC185
Rab1

Rab1

cis
medial
Rab6

Rab6

trans
Rab6

Sinka et al., 2008 J. Cell. Biol. 183, 607.
Hayes et al., 2009 Mol. Biol. Cell 20, 209.

Molecular level

Arl1

Cell level

Golgin structure

Maintenance of Golgi stack structure

Specific interactions between
Golgin and Rab/Arf

Localization of Rab/Arf on
different Golgi compartments

Endocytosis of toxin -> Drug delivery
Clathrin movie
by Allison
Bruce, Harvard
http://www.hms.ha
http://wwwhms harvard.edu/news/cl
athrin/David S. Goodsell, Scripps Institute, http://www.scripps.edu/mb/goodsell/Human GGA: a new class of adaptor proteins
GAT
Clathrin
terminal domain① VHS

Clathrin

50 nm

GAE

③

ARF-GTP
M6PR

②
MPR

N-GAT

Autoinhibition

33 nm

Accessory protein

clathrin box
Hinge region

Competition
TGN membranewith AP-1

Lumen① Shiba et al. Nature 415,937-941, 2002
cargo
g protein
ff , vol. 5,, 437-448,, 2004
Shiba et al.,, Traffic,② Shiba et al., Nature Structural Biology, 10 386-393, 2003
Shiba et al., J. Biol. Chem. 279, 7105-11279, 2004
Kawasaki et al.,, Genes to Cells,, 10,, 639–654,, 2005
Yogosawa et al., BBRC, 350, 82-90, 2006
③ Nogi et al. Nature Structural Biology, 9, 527-531, 2002
Inoue et al. Traffic, 8, 904-913, 2007

Ubiquitin (Ub): ubiquitous
protein modifier with only 76
amino acid residues,
residues 27000
papers published to dateCan connect Ub to another Ubusing
g 7 ((now 8)) different ways
y

K63

K48

How are specific linkages
recognizedgfor signaling?
g
g

N

K29
K27
K33

K6
K11

C

7 lysines of ubiquitin

Polyubiquitin: linkage specificity vs. function
Main functions
Ub

Ub

Ub
Ub

Ub

DNA repair

Ub

K63

Ub

N

K29
K27
K33

P t i degradation
Proteindd ti

ProteinUb

K6

K48-linked
K48
linkedUb

Poly-ubiquitylation

K48

Endocytosis

P t iProteinMono-ubiquitylation

K63-linked

DNA transcription
Vesicle transport

LinearDNA transcription

ProteinK11

Ub Ub Ub Ub
C

7 lysines of ubiquitin

ProteinLinear-ubiquitylation and NEMO (Rahighi, et al., Wakatsuki, Dikic, Cell 2009;
Tokunaga et al., Iwai, Nature Cell Biol., 2009)

7 lysines, K48, K63 and Linear

Lange
ett al.,l
Science2008
Taken from Dikic, Wakatsuki, Walter, Nat. Rev. Mol. Cell Biol., in press

Ubiquitinq
is a multifaceted p
protein

Ashura, Kofuku-Ji
TempleFinding NEMO

Structural Biology Research Center
Photon Factory, IMSS, KEK, Tsukuba,
Japan

Goethe University, Frankfurt, Germany
MRC, Cambridge, UKTeams behind the Ubiquitin/NEMO project

Simin Rahighi

Ivan Dikic

Fumiyo Ikeda

Ivan Dikic & Fumiyo Ikeda, Inst. of Biochemistry, Goethe
Univ Med School, Frankfurt, Germany
M kS
Mark
Schmidt
h id Supprian,S
i
Germany
G
Felix Randow, MRC, Cambridge, UKDavid Komander
Komander, MRC,MRC Cambridge
Cambridge, UK
Simin Rahighi, Masato Kawasaki, Nobuhiro Suzuki, Tamami
Uejima,j, RyuichiyKato,, SBRC,, KEK-PF,, Tsukuba,, Japanp

Ubiquitin signals in NF-κB activation
Multiple agonists

1st Ub signal
Lys63 chain

2nd Ub signal
TAK1

IKKα
IKKβ

NEMO

NEMO: NF-kB essential
modifier
3rd Ub signal
Lys48 chain

Immune response,
inflammation and cell divisioninflammation,

I-κB
p65
65

DNA transcription

p50
50

UBAN - Ub binding domains present in
ABIN and NEMO proteins
58
1

96
HLX1

187
CC1

202

243 250 285
CC2
HLX2
250

314 336
LZ

UBAN
289

338

390

410
ZF

412

Structure of ubiquitin binding domain of NEMO
N

180.0˚ rotation
N

N

N

N

N

180.0˚ rotation

Pro292
N
N

Janus
C C

NEMO-UBANCCHow is linear ubiquitin chain recognized by NEMO?
━ How does NEMO differentiate linear vs. K63 linkage ━━ How does Ub binding activates ＩＫＫ? ━
58

96
HLX1

1

187

202

CC1

243 250 285
CC2
HLX2
250

314 336
LZ

UBAN

390

410
ZF

412

338

289

IKKα
(C)

(D)

IKKβ

(A)

(B)

Supplementary Figure 1. Model of the overall NEMO structure based on the four structures:(A) Rushe M., et al., Structure 16, 798-808, May 2008
(B) Modified from Bagneris C.,
C et al.,al Molecular cell 30,
30 620-631,
620 631 June 6,
6 2008(C) UBAN domain: Current work(D) Cordier F., et al., J.Mol.Biol. 377 (5): 1419-1432, April 2008???

K
Substrate

Ubiquitin binding domain of NEMO (UBAN)
specifically recognizes linear-diubiquitin
linear diubiquitin
58
1

96
HLX1

187
CC1

202

243 250 285
CC2
HLX2
250

314 336
LZ

UBAN

390

410
ZF

412

338

289

ＳＰＲ measurement of
NEMOUBAN/diubiquitin
dissociation constant:
1.6 μM

Structure of ubiquitin binding domain of NEMON

UbdistalN

Ubdistal

Ubproximal

N

UbdistalN

C

Ubproximal
Side view

C

Ubproximal

Ubdistal

Ubproximal
Top viewC

UBAN dimer in complex with two linear diubiquitin molecules

Distal ubiquitin interaction
b

Oε
Oε

E289

N N

P292

H68

V293

G47
L8

L294

I44

A296

R42

Q297
V70

I300

L71

Y301

Oη

O

R72

Distal site

N

Nη
Nη

K302
Oδ
Oδ

L73
N

R74

Proximal
site

Nη
Nη

Oε
Oε

D304
F305
A307
E308
R309

R312

R309
R312

Proximal ubiquitin interaction
d

Oε
Oε

E16
Nε

Q2

Nη
Oε

Nη

N

E64
I 13

R312
Oε
Oε
Oε
Oε

Oγ

T12

Oε
Oε

G10

Proximal
site

E313
E317

T66
K6

R309

Nη

T14
F4

Nη

E320

Cornell Group’s models based on
NMR titration, ITC, mutagenesis

Distal Ub

Proximal Ub

K63-linked diubiquitin

Linear diubiquitin

Distal Ub
Proximal Ub
Lo et al., Mol Cell,
EPUB 29 Jan, 2009

K63 and NEMO: 2:1 structure by Fukai’sgroup,
gp, FEBS Letters,, Sep,p, 2009
NN

Ubdistal

Ubdistal

Ubproximal

Ubproximal

C
Rahighi etC al., Cell,
March 2009

Yoshimura et al., FEBS
Lett, September 2009Lo et al., Molecular Cell,
January 2009

Wu’s group
UBAN:diUb = 2:1
Multi Angle Light Scattering (MALS) and ITC,
ITC: later byy Rittinger
g and KEK

Jan 29, 2009, online publication

Stoichiometry controversy, continued: sedimentation
velocity analysis by Rittinger’s
Rittinger s group
group, Ivins et alal,
Biochemistry J, 421 243–251, 2009

2:1

2:2

CoZi
C
Zi andd di
di-Ub
Ub iinteracttt tto formf
C Zidi
CoZidimer/diubiquitin/di bi iti andd att hihigherh ubiquitin
bi itiratios (CoZi dimer) / diubiquitin 2. Best fit C(s) functions for sedimentation
velocity analysis of CoZi/Ub2 interaction(s).

Isothermal calorimetry shows both 2:1 & 2:2

Purely proximal E313A NEMO
mutation
Di l site
Distal
i
wt

V293A
Y301A
K302A

F305A

R309A
R312A
E313A

E313A

α-AU1
37kD

25kD

Proximal siteLinear-diUb recognition of NEMO is critical for
LPS-induced NF-κB activation in vivo
Dominant negative: one WT and one
mutant in the coiled-coilcoiled coil dimer

100

κB:G
GFP (%)

med
LPS

50

0
control

Heterodimers formed byintroducing NEMO mutant
alleles into wild type B cells

WT

V293A
Y301A
K302A

F305A

Nemo

R309A
R312A
F313A

NEMO is a long alpha helical dimer
How does Ub binding activate IKKα and IKKβ?
Substrate

K

IKKα

(D)

IKKβ

(A)

(B)
((C))

A) Rushe M
M., et alal., Structure (2008)
B) Bagneris C., et al., Mol. Cell (2008)
C) Current workD) Cordier F., et al., J. Mol. Biol. (2008)

K
SubstrateConformational change in NEMO upon binding tolinear diubiquitin chains
N
N
Glu289Glu32090°

Apo-form

CC
Overlapped90°

UBAN/Ub2 complex

Binding of two linear ubiquitin chains unwinds,
slightly the NEMO
slightly,
NEMO-UBANUBAN domain⇒ may lead to structural change of the entire NEMO━

RahigiR
hi i & Ik
Ikeda
d ett al.,l Cell,C ll online
li publication:
bli ti
March
M
h 20,
20 2009
In collaboration with Ivan Dikic, Goethe Univ. Frankfurt,David Komander & Felix Randow, MRC Cambridge

Specific interaction of NEMO-UBAN
to linear vs.
vs K63-linked diubiquitin linkages
NEMO-UBAN

Ubdistal
G75
E16
G76
R309

Q2A mutation inproximal Ub
disrupts NEMO
binding

M1
K63

Q2

R312
α-AU1

Ponceau S

Ubproximal

Green: NEMO & Linear Ub2
Gold: NEMO & Lys63-linked Ub2 (model)

Variety of K63/linear diUb structures
UbdistalFabNEMOFree (Rohaim)

Free (Komander)

Ubproximal
AMSH-LPK63 diUb (Free K63): Komander et al., EMBO Reports, 2009
K63 diUb (Fab): Newton et al., Cell, 2008
K63 diUb (AMSH(AMSH-LP):
LP): Sato et alal., Nature,
Nature 2008
Linear diUb (NEMO): Rahighi et al., Cell, 2009
Linear diUb: Rohaim et al. (unpublished)K48 diubiquitin
NMR by Varadan et al. Mol Cell, 2005

Linear diubiquitin
X-rayy byy Rahighig et al.,, Cell,, 2009

No contact

K63 diubiquitin
Model by Sims & Cohen, Mol Cell 2009
X-ray
X
ray by Sato et al.,al EMBO J.,
J advance
online publication 18 June 2009

Double sided UIM
X-ray by Hirano et al., NSMB, 2005

Mutations in NEMO in patients suffering fromectodermalt dl dysplasiadl i with
ith immunodeficiency
id fi iNEMOUBAN

DistalUbiquitin

R72
L73
R74

D304N
E308A

Q2
F4
E64

Proximal
Ubi itiUbiquitin

R312Q

EDA mutants of human
NEMO: D311N, E315A
and R319Q

Model of Ub signaling in the NF-κB pathway
New paradigm in ubiquitin signaling in the NF
NF-κBκB
pathway: linear ubiquitin recognition
Plasma membrane

LUBAC linear
Ub ligase
Degradation of IκBαLiner polyUb
IKK complexes
Into nucleus

NucleusA model for the NEMO linear Ubiquitin binding
and ubiquitylation

Complex patterns of posttranslational
modifications of IKK complex
From Perkins,
Perkins Oncogene,Oncogene
2006

Drug
g design
g targetsgTargetinggg ubiquitinqitself withubistatins: Verma et al, R.W.
King, Science, 2004

Dikic, Wakatsuki, Walter, Nat. Rev. Mol. Cell Biol., in press

Plethora ofubiquitin chains
may lead tonumerous
biological signals⇒ New ubiquitin
world

Lys 48/Lys 29
Kim et al, JBC 2007

Linear!
Tokunaga, Iwai et al.,
NCB, 2009

Tatham et al, NCB 2008

Previously:
y 7x7x7x7x7 = 16807
With linear: 8x8x8x8x8 = 32768
Taken from Ikeda & Dikic, EMBO Reports, 9, 536-542 (2008)

Comparison of ERL, SASE-FEL and XFEL-O

ERLXFEL-OSASE FEL
SASE-FEL

averagebrilliancepeakbrilliance

repetition
rate (Hz)

coherent
fractionbunchwidth(ps)

# of
BLs

Remark

~1023

~1026

1.3G

~20%

0.1~1

~30

Non-perturbed
measurement

~1027

~1033

~1M

100%

1

few

Single mode
FEL

SASEFEL~1022~24~1033

100~10K

100%

0.1

~1

One-shotmeasurement

3rd-SR~1020~21

~1022

~30

Non-perturbed
measurement

ERL
XFEL-O（Option)

~500M

0.1%

10~100(brilliance : photons/mm2/mrad2/0.1%/s @ 10 keV)Radiation damage: photoelectrons escape sideways.
Polarization direction of the SR

1 micron beam

2 micron beamWill reduced radiation damage occur with very small crystals?
Colin Nave and Mark A. Hill, J. Synch. Rad. (2005). 12, 299–303

New Ubiquitin World with Accelerator Technologies
Neutron reflectivity
GI‐SANS & GI‐SAXS

Analysis of receptors upon
agonist binding

NF kB pathway
NF‐kB

Macroscopic structural changes ⇔ function
Hierarchicalstructure ofubiquitinrecognition

Substrate

K

IKKα

(D)

IKKβ

(A)

(B)
(C)

K
Substrate

SAXS
Protein Crystallography

Time resolvedTi
l d solution
l ti structuralt t l analyses
lof posttranslational modifications & complex
formation

Accelerate developments of drugs
Imaging localization of protein
complexes with high resolution andcontrast

KEK‐X

SR X‐ray tomography

Structural studies of polyubiquitin synthesis & recognition
In vivo analysis of drug delivery and signal transduction

Transient structural dynamics of signal transduction
complexes: Small Angle X-ray Scattering (SAXS) with next
generationi 2D detectorsdandd iink-jet
k j technologies
h l i
Rapid mixing of components using
ink-jet technologies
Linearly ubiquitylated
substrate protein

IKKα・IKKβ・NEMO complex

Fastt time-resolvedFti
l d SAXS
using next generation 2D
detectors (SOI, APD, etc.)

Ultra hiUlt
highhbrilliance beamfrom KEK-X

Substrate

K

IKKα

(D)

IKKβ

(A)

(B)
(C)

K
Substrate

Elucidation of IKK activation
mechanism through interaction
between NEMO and linear ubiquitin
chainsSolution structure of IKKα・
IKKβ・NEMO and linearlyubiquitilated substrates

How large
g is the pprotein universe?
• How many genes?
– MetaM t genomes --- J.
J C
Craig
i VenterV t Institute's
I tit t 'Global Ocean Sampling Expedition– Human microbiomes (Gill, et.al. David Relman,
Stanford, Science 2006)– Emerging infectious diseases• Splicing variants – exon: cut and paste• Non-coding RNA: largely uncharted• Protein-protein, protein-carbohydrate,
protein-lipid
proteinlipid, protein-nucleic
protein nucleic acid interactions• Posttranslational modifications

PSI:Biology
gy of NIGMS/NIH
PSI Biology large centers: Applications by Oct 28th

http://www.nigms.nih.gov/Initiatives/PSI
1. Concept Clearance: High-Throughput Structural Biology
http://www.nigms.nih.gov/News/Reports/council_concept_clearance_2009.htm
2 Recommendations for the Future of the PSI
2.
<http://www.nigms.nih.gov/News/Reports/PSIAC_Future_2009.htm> (PSI
Advisory Committee)
3 Report of the Future Structural Genomics Initiatives Meeting3.
<http://www.nigms.nih.gov/News/Reports/FutureSGMeeting102008.htm>
4. Annual PSI Advisory Committee Meeting Report<http://www.nigms.nih.gov/News/Reports/PSIAC2008.htm>
http://www.nigms.nih.gov/News/Reports/PSIAC2008.htm
5. PSI Steering Committee Meeting Report<http://www.nigms.nih.gov/News/Reports/psi_steering_2008.htm>

Global Ocean Sampling Expedition• Sorcerer II of Craig Ventor Institute• GOS analysis identifiedfsome 6.12 million
proteins from 7.7 million newly discovered
sequencesHuman microbiomes• WithinWithi ththe bbody
d off a hhealthylth adult,
d lt
microbial cells are estimated tooutnumber human cells by a factorof ten to one.
• Human ggut: 1000 microbiomes,,
1x1014 cells, 1 kg per person, but
so far only 40 species have beencharacterized.
characterized• The NIH Roadmap Initiative now
includes a Human Microbiome
Project(HMP,http://nihroadmap.nih.gov/hmp))• JCSG (NIGMS PSI) collaboration
on structures

Structural Biology Research Center, Photon Factory, KEK
Soichi Wakatsuki

Professor, Director of Structural Biology Research Center

Structural Biology
Ryuchi Kato (Associate Prof.)
Masato Kawasaki (Assist.
(Assist Prof
Prof.））
Kentaro Ihara （Post Doc Fellow）
Norio Kudo （Post Doc Fellow）
Nobuhiro Suzuki (Post Doc Fellow)
Hisayoshi Makio (Post Doc Fellow)
Seiji Okazaki (Post Doc Fellow)
Tamami Uejima（Researcher）
j
Tamie Aoki（Lab Technician）
Tomoya Honda （Lab Technician）
Ex members:
Tadashi Satoh （Post Doc Fellow）+++

Beamline & Robotics
Noriyuki Igarashi （Assoc. Prof.） BL6A, BL17
Masashiko Hiraki （Lecturer）
Robotics
Naohiro Matsugaki （Assist. Prof.） NW12, BL1
Yusuke Yamada （Assist. Prof.）
NE3
Leonard Chavas (Assist.Prof.)
Kumiko Sasajima（System Engineer）
Shokei Watanabe （Mechanical Engineer）
Nobuo Honda（System（ yEngineer）
g）
Kazutaka Demura（Beamline Assistant）
Takayuki Kubota （Beamline Assistant）
Rei Tanabe (Beamline Assistant)
Shinji Naito (Beamline Assistant)

The Graduate University for Advanced Studies
Simin Rahighi (D3), Ahmed Rohaim (D2), Kensuke), Ken Ohkubo (M2)
( )Nakamura ((M2),

Secretaries
Gin Ishikawa
Tomoko Zeniya
Ritsuko Ebisawa
Reiko Sukegawa

Internship Students: Atsuko Tsuchiya (Kyoto Univ. M1), Ryunosuke Nakatani (Nagaoka Univ. of Tech. B4)

AcknowledgementsgSPring-8S
g 8 & JASRIS
Masaki Yamamoto
Takashi Kumasaka
Hokkaido
H
kk id University
U iit
Isao Tanaka
Kyoto University
Kunio Miki
Osaka University
Atsushi NakagawagMamoru Suzuki

Financiala c a Supports
Suppo sDevelopment of Systems andTechnology for Advanced
Measurement and Analysis(JST)
Protein 3000 Project (MEXT)Grand-in-Aid for Young
Scientists ((B)) 18770098(MEXT)
Target Protein Research
Program (MEXT)

Nagoya University
Nobuhisa Watanabe

Astellas Pharmaceutical Inc.