2.5 GeV PF

Science with Future Hard X-ray Sources: Exploration of

Protein Universe

ICFA Future Light Source Workshop, 2 March 2010, Stanford

6.5GeV PF-AR

7 GeV & 4 GeV KEK-B ring (⇒ Super KEK-B & KEK-X )

cERLERL

Soichi WakatsukiPhoton FactoryIMSS, KEK

Role of Structural Biology

Medicine, drug design(industrial applications)

Biology(Basic research)

DNA→mRNA→Genome

Molecular machines（ribosomes, enzymes）Information network

Signal transduction

Atomic resolution analysis○Protein structure and dynamics○Interactions between molecular machines

Protein transport

Proteins

Ribosome

Translation

Transcription

Nobel Award in Chemistry, 2009

• Ribosome structures• Prof. Ada Yonath was a user of the Weissenberg Camera

with large IPs developed by Prof. Sakabe, Photon Factory for 10 years from 1987– Symposium of Target Protein Resarch Program March in Tokyo,

on 5th, 2010– Photon Factory Symposium in Tsukuba on March 9, 2010

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

Harms et al., Cell, 107, 679-88 (2001)

http://www.weizmann.ac.il/sb/faculty_pages/Yonath/HarmsCELL2001.pdf�http://www.weizmann.ac.il/sb/faculty_pages/Yonath/HarmsCELL2001.pdf�http://www.weizmann.ac.il/sb/faculty_pages/Yonath/HarmsCELL2001.pdf�

The Worldwide Protein Data Bank (wwPDB) http://www.wwpdb.org/index.html

David S. Goodsell, Scripps Institute

>60,000 entries and growing!

How large is the protein universe?• How many genes?

– Meta genomes --- J. Craig Venter Institute's 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, protein-nucleic acid interactions

• Posttranslational modifications

From atomic structures toIn situ observation of biological nano machines for cell/tissue level understanding

Understanding of self-organizing biomolecularnano machines using SR X-ray nano beam

Signal transduction through membrane

proteins in the lipid raftFlagellin formation

Namba and coworkers

Extraordinarily large Vault complexTanaka et al. (2009) Science

67nmLipid raft

What do we want to know?• Large complexes: real time, real place

(organelle/cell/tissue), at atomic resolution• Dynamics of multicomponent complexes:

hierarchical structure• Imaging: 5 nm or better spatial resolution to

discriminate inside/outside of membranes.• Imaging with chemical speciation is not sufficient:

spectroscopy required for certain components which include metal proteins

• Structural changes at the membrane surface: lipid rafts with membrane protein complexes: can time-resolved GISAXS be the solution?

Figure by David S. Goodsell, Scripps Institute

View of a eukaryotic cell

COLORS: proteins in blue, ribosomes in magenta, DNA and RNA in red and orange, lipids in yellow, and carbohydrates in green.

http://www.scripps.edu/mb/goodsell/

•Red boxes: about ~50 nm by ~100 nm.•Imaging needs better than 5 nm resolution.•COHERENT BEAM: X-ray photon correlation spectroscopy (XPCS) to study the dynamics?

Protein-protein interaction network

Taken from Rua et al., Nature 2005

Nucleus

Endoplasmic Reticulum

TGN

Plasma Membrane

Protein glycosylation

Golgi apparatus

Endocyticpathway

Autophagic pathway

Lysosome

Clathrin Coated Vesicle Early Endosome

Late Endosome

Autolysosome

Secretory Vesicles

Systems Structural BiologyPosttranslational modification and transport

GGA

Melanosome

Transport in nueronsFrom N. Hirokawa, J. of Neuroscience, 2006

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

Double headed kinesin

1~2 µm/sec8 nm step

~0.5 µm

N. Hirokawa, Univ of Tokyo Med School

Endocytosis of toxin -> Drug delivery

David S. Goodsell, Scripps Institute, http://www.scripps.edu/mb/goodsell/

Clathrin movie by Allison Bruce, Harvard http://www.hms.harvard.edu/news/clathrin/

TGN membrane

GAE

Hinge region

GAT

Accessory protein

Lumen

N-GAT

Clathrin terminal domain

Clathrin

clathrin box

cargo protein

M6PR

VHS

ARF-GTP

MPR

①

②

③

Human GGA: a new class of adaptor proteins

Auto-inhibition

Competition with AP-1

① Shiba et al. Nature 415,937-941, 2002Shiba et al., Traffic, vol. 5, 437-448, 2004

② Shiba et al., Nature Structural Biology, 10 386-393, 2003Shiba et al., J. Biol. Chem. 279, 7105-11279, 2004Kawasaki et al., Genes to Cells, 10, 639–654, 2005Yogosawa et al., BBRC, 350, 82-90, 2006

③ Nogi et al. Nature Structural Biology, 9, 527-531, 2002Inoue et al. Traffic, 8, 904-913, 2007

33 nm

50 nm

EGFP-Arf6 MKLP1 β-tubulin

Cytokinesis needs lots of new membranes

Tubulin

Cleavage furrow

midbody

Colocalization of MKLP1 & Arf6 in midbody

Cytokinesis seen with EGFP-FIP3 and mCherry-Rab11

微小管microtubules

MKLP1

Exocyst complex

Cyk4

Arf6 Arf6

細胞膜 Plasma membrane

分裂溝Cleavage furrow

+ -+-

+-

Rab11

FIP3Rab11

Fusion

BAR domain

Homology

N-terminal C-terminal

1 108 121 321 342

80%Arfaptin1

Arfaptin2

BAR (Bin/Amphiphycin/Rvs)

domain: a module that is involved

in dimerization, membrane

binding and curvature-sensing.

Arfaptin/POR function in membrane trafficking

Taken from Wang et al., Structure, 2008

Time lapse images

Arfaptin2 & Arl1

Arl1/Arfaptin tubulationmovie part 1 by Yuko

Tsukihara (Meta Corporation Japan)

Arl1/Arfaptin tubulationmovie part 2 by Yuko

Tsukihara (Meta Corporation Japan)

K6

K11

K27K29

K33

K48

K63

N

C

K6

K11

K27K29

K33

K48

K63

N

C

Ubiquitin (Ub): ubiquitous protein modifier with only 76 amino acid residues, 27000 papers published to date

Can connect Ub to another Ub using 7 (now 8) different ways

How are specific linkages recognized for signaling?

7 lysines of ubiquitin

K6

K11

K27K29

K33

K48

K63

N

C

K6

K11

K27K29

K33

K48

K63

N

C

Protein

Ub

Mono-ubiquitylation

Poly-ubiquitylation

Endocytosis

DNA repair

Protein

Ub

Protein

Ub

Protein degradationUb

Ub

Ub

UbUb

Ub

K48-linked

K63-linked

Main functions

7 lysines of ubiquitin Protein

Ub DNA transcriptionUb Ub Ub Linear

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

Polyubiquitin: linkage specificity vs. function

DNA transcriptionVesicle transport

7 lysines, K48, K63 and Linear

Lange et al.,

Science 2008

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

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

ＳＰＲ measurement of NEMO-UBAN/diubiquitin dissociation constant: 1.6 µM

CC1 HLX2 CC2 LZ ZF58 96 187 202 243 250 314 336 390 410

1 412HLX1

250 338UBAN

289

285

Model of Ub signaling in the NF-κB pathwayNew paradigm in ubiquitin signaling in the NF-κBpathway: linear ubiquitin recognition

Plasma membrane

Degradation of IκBα

Nucleus

Into nucleus

Liner polyUbIKK complexes

LUBAC linear Ub ligase

Immune response,

inflammation, apoptosis, cancer etc.

Cytokines

N

CC

N

Ubdistal Ubdistal

UbproximalUbproximal

Rahighi et al., Cell, March 2009 2:2

Lo et al., Molecular Cell, January 2009 2:1

Yoshimura et al., FEBS Lett, September 2009 2:1

Crystallographic controversy NEMO: diUbiquitin =2:1 vs 2:2NEEDS MUCH BETTER METHOD TO RESOLVE THIS!

(A) (B)(D)

IKKα

IKKβ

Substrate

Substrate

K

K

(C)

NEMO is a long alpha helical dimer

How does Ub binding activate IKKα and IKKβ?

A) Rushe M., et al., Structure (2008)

B) Bagneris C., et al., Mol. Cell (2008)

C) Current work, Rahighi, Ikeda et al., S. Wakatsuki, Ivan Dikic, Cell (2009)

D) Cordier F., et al., J. Mol. Biol. (2008)

Eli Lilly and Companynow has the structures of IKKα and IKKβ!!!

Plethora of ubiquitin chains may lead to

numerous biological signals ⇒ New ubiquitin world

Lys 48/Lys 29

Taken from Ikeda & Dikic, EMBO Reports, 9, 536-542 (2008)

Linear!

Tokunaga, Iwai et al., NCB, 2009

Kim et al, JBC 2007

Tatham et al, NCB 2008

Previously: 7x7x7x7x7 = 16807

With linear: 8x8x8x8x8 = 32768

(A) (B)(D)

IKKα

IKKβ

Substrate

Substrate

K

K

(C)

Time resolved solution structural analyses of posttranslational modifications & complex formation

Accelerate developments of drugs

Imaging localization of protein complexes with high resolution and contrast

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

Analysis of receptors upon agonist binding

Macroscopic structural changes ⇔ function

SAXS

Protein CrystallographyRahighi et al., Cell 2009

SR X-ray tomographyFigure by C. Larabell

Neutron reflectivityGI-SANS & GI-SAXS

NF-kB pathway

New Ubiquitin World with Accelerator Technologies

KEK-X

Hierarchical structure of

ubiquitin recognition

Roadmap of protein crystallography beamlines in JapanExamples A: membrane proteins, B: enzymes without heavy atom lables

& C: virus crystals

Size

of c

ryst

al

> 200μm

1μm

B

Size

of c

ryst

al

A

CAR-NE3A

BL-5AAR-NW12A

BL-17A

BL-1A

PF

BL-6A

SAGABL-7

SPring-8

BL44XU（Osaka Univ）

BL41XU

BL32XU

BL38B1 BL26B1/2

（RIKEN）

•Yearly budget: 5.5 billion yen, US$ 44.5M (includes 30% overhead)•43 teams selected on 15 June 2007 & 2 new teams joined in 2009•So far, papers in Nature 13, Science 2, Cell 7•KEK: Vesicle transport & Structural Analysis Core

Structural Analysis Core

Microfocus (SP8) & long λ SAD(PF)

Protein Production Core

Informatics Core

Functional Control Core

(140K compounds Chem. Library)

Medical importance/relevance

Fundamental Biology

Food and environmentTargets:

Target Protein Project (5 years: 2007-2011)

5 yr term 7 6 5

3 yr term 5 4 6

5 yr 1 1 (X-ray) 1 13 yr 3 2 (NMR) 0 1

http://www.tanpaku.org/e_index.php

Extremely large complex, Vault(Tsukihara Group, Science 2009)

• Structure determined at 3.5 Å reslution with 39-fold symmetry• MVP(major vault protein) alone is 99kDa x 78 copies = 7.7 MDa• SPring-8 BL44XU Osaka 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 0.15 mm x0.03 mm, and belongs to the space group C2, with cell dimensions of a = 702.2 Å, b = 383.8 Å, c = 598.5 Å, and β = 124.7°.

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

How many unit cells in micro/nano crystals do we need for structure determination?

Unit cell dimension

Crystal size (micro cube)

30Å 40Å 50Å 100Å 200Å 300Å 500Å 800Å

300 1.0E+15 4.0E+14 2.0E+14 3.0E+13 3.0E+12 1.0E+12 2.0E+11 5.0E+10

200 3.0E+14 1.0E+14 6.0E+13 8.0E+12 1.0E+12 3.0E+11 6.0E+10 2.0E+10

100 4.0E+13 2.0E+13 8.0E+12 1.0E+12 1.0E+11 4.0E+10 8.0E+09 2.0E+09

50 5.0E+12 2.0E+12 1.0E+12 1.0E+11 2.0E+10 5.0E+09 1.0E+09 2.0E+08

30 1.0E+12 4.0E+11 2.0E+11 3.0E+10 3.0E+09 1.0E+09 2.0E+08 5.0E+07

20 3.0E+11 1.0E+11 6.0E+10 8.0E+09 1.0E+09 3.0E+08 6.0E+07 2.0E+07

10 4.0E+10 2.0E+10 8.0E+09 1.0E+09 1.0E+08 4.0E+07 8.0E+06 2.0E+06

5 5.0E+09 2.0E+09 1.0E+09 1.0E+08 2.0E+07 5.0E+06 1.0E+06 2.0E+05

4 2.0E+09 1.0E+09 5.0E+08 6.0E+07 8.0E+06 2.0E+06 5.0E+05 1.0E+05

3 1.0E+09 4.0E+08 2.0E+08 3.0E+07 3.0E+06 1.0E+06 2.0E+05 5.0E+04

2 3.0E+08 1.0E+08 6.0E+07 8.0E+06 1.0E+06 3.0E+05 6.0E+04 2.0E+04

1 4.0E+07 2.0E+07 8.0E+06 1.0E+06 1.0E+05 4.0E+04 8.0E+03 2.0E+03

Numbers of “cubic shape” unit cells in cubic shape crystals. Colored according to the number of unit cells contained in crystals: more than 108 copies in green, 107

pink, & 106 copies yellow. （by Masaki Yamamoto, Harima RIKEN/SPring-8）

Micro crystals of cypovirus polyhedraby Peter Metcalf et al.

• Structure at 2 Å resolution(Peter Metcalf group, Nature 2007, data collected at Swiss Light Source, presented in AsCA 2009, Beijing)

• Very robust crystals：dissolve only above pH10

• a=b=c= 103 Å, １ micron cube crystal ⇒ contains 106unit cells ⇒ 100 repeats along each axis

Laue function of micro crystalsLimit 10 x 10 x 10 = 1000 ?

Laue

func

tion

For Na = 10

• Intense micro/submicro beam with low divergence (0.1 mrad or lower in H direction)

• Stability ~10-3: with the next generation PADs, each reflection might see the beam for 1~10 msec

Target Protein Research Project (MEXT) Microbeam Beamline BL32XU

Achieved beam size （2009/11/27） by SPRing-8

-3 -2 -1 0 1 2 30.0

0.2

0.4

0.6

0.8

1.0

Inte

nsity

/ ar

b. u

nit

Position / um-3 -2 -1 0 1 2 3

0.0

0.2

0.4

0.6

0.8

1.0

Inte

nsity

/ ar

rb. u

nit

Position / um

Focused photon flux : 5.7x1010 photons/secDivergence: 0.5 mrad (H) x 0.009 mrad (V)

Horizontal beam profile Vertical beam profile

FWHM0.69 µm

FWHM0.78 µm

SP8

Facility Beamline Vert(µm)

Width(µm)

Flux (phs/sec)Flux density

(phs/sec/µm2)

APS 23-ID-B ４ µmφ 4.0E+10 3.2E+09ESRF ID23-2 7.5 5 4.0E+11 1.1E+10

SLS X06SA 25 5 1.0E+12 8.0E+09

X10SA 50 5 1.0E+12 4.0E+09

SPring-8 BL32XU 1.1 1.0 6.2E+10 5.6E+1019.3 7.2 6.7E+12 4.8E+10

http://biosync.rcsb.org/international.html(as of Nov 20th, 2009）

Microfocus beamlines around the world

SP8

http://biosync.rcsb.org/international.html�

Crystal size and diffracting powerTaken from Acta Cryst. (2008), D64, 158-166

◆ Protein crystals

□ Organic small molecule crystals

cryst32

cell000 )/( VVFS ××= λ

※2Å data collected from a 2µm lysozyme crystal

SPring-8 BL32XU

Diffracting power

SP8

20 µmLysozyme crystal

Sample Thermolysin 1 Thermolysin 2

Laser trapping(λ = 1064nm)

760mW, 1min.

none 760mW, 1min.

none

Data Collection Statistics

Resolution (Å) 50 - 2.08 (2.15 – 2.08)

Space group P6122

Cell dim: a,c (Å) 92.7, 129.3 92.8, 129.3 92.6, 129.4 92.7, 129.3

Mosaicity (º) 0.127 0.100 0.168 0.174

I 403 (153) 460 (179) 539 (196) 725 (263)

I / σ( I ) 44.7 (17.9) 45.9 (18.6) 46.0 (17.6) 52.9 (21.5)Multipicity 10.4 (9.9) 10.4 (9.9) 10.5 (10.0) 10.4 (10.1)

Comp. (%) 99.6 (99.3) 99.5 (98.6) 98.9 (96.6) 99.0 (97.8)

R merge (%) 8.7 (15.6) 8.6 (14.8) 8.1 (15.2) 7.6 (14.1)

Structure Refinement Statistics

R factor (%) 17.8 18.0 17.5 17.6

R free (%) 21.1 22.9 20.6 21.3

Ave. B (Å2) 9.88 9.43 9.87 10.26

R.m.s. dev. (Å) 0.031 0.033

50µm

Laser TrappedPoint760mW, 1min

X-ray ExposedPoints

1064nmYAG Laser

microscope

1064nmYAG Laser

XYZManipulator

XYZManipulator

Lensed FiberProbes

CrystalSolution

3D manipulation of lysozyme crystal（Laser power = 25mW x 2）

Test for damage on sample

Development of Micro-crystal handling with laser tweezers being developed by SPring-8

Crystal tweezers with fiber laser optics

50µm

SP8

PF-AR NW14A: ERATO Project (~2009): Dynamics studies with 100 ps time resolution for innovation in materials and biological sciences

Tokyo Institute of Technology, ERATO (JST) S. Koshihara、KEK・PF Shin-ichi Adachi

Laser driven shock waves through CdS single crystal

Dynamics of ligand migration in protein crystal

Solution photo reaction

Laser induced spin crossover

reaction

39

AcetylcholinesteraseProf. Joel Sussman et al.

Weizmann Institute, Israel50,000 hydrolysis reactions per second, i.e. 20 µsec/event

So far, it has not been possible to observe the protein breezing motion in the crystal despite numerous efforts including Laue experiments. -> Need to do time-resolved SAXS at one micro sec or faster time resolution with light cleavable cage compound.

http://www.weizmann.ac.il/sb/faculty_pages/Sussman/images/big_wh_ray01.gif�

Single molecule structure determination with hard x-ray FEL

• Radiation damage: avoidable if X-ray photons are as short as several fsec (?)

• How to average coherent diffraction images (fundamentally different from crystallography where averaging is done by the crystal). Determination of orientation of the molecule crucial

• How many water molecules are allowed to be attached to the proteins?

• How many lipids need to be there for membrane proteins to stay in the active form when electron-sprayed?

• Conformations of the single molecules (complexes) in the beam have to be the same for all the images to be used for structure determination – rather difficult

Dikic, Wakatsuki, Walter, Nature Review Molecular Cell Biology, 2009

Drug design targets: can we freeze the complexes?

Targeting ubiquitin itself with ubistatins: Verma et al, R.W. King, Science, 2004

Ultimate storage ring and/or ERL• Ultimate protein crystallography: nano crystals

with million or fewer copies

• Coherent diffraction of organelles/cells and other large structures such as chromosomes

• X-ray microscopy/tomography: finding new organelles or distribution thereof.

• XPCS of subcellular regions for dynamics• Grazing incidence SAXS to study structure and

dynamics of membrane proteins/lipid rafts

Understanding hierarchical structure/function using CDI, crystallography, SAXS, GISAXS, EM, NMR, etc.

Small Angle X-ray Scattering

Diffraction image Reconstructed yeast cell(by C. Jacobsen, Stony Brook)

Imaging needs 5 nm or better resolution to see inside/outside of membranes

Signature with metal incorporation (eg. 3-iodo-L-tyrosine) into different proteins

Adapter protein GGA Clathrine

Motor protein on microtubule

Ribosome

Preliminary Design of 5GeV ERL

XFEL-O

KEK-X Project with the upgrade of KEKB to Super KEKBLER

PF-AR 7 GeVinjection and top-up operation?

LER

HER

HER

Preliminary plan ofLER West-Symmetry Point Experimental hall

150m x 30m = 4500 m2

ID26

ID28

ID30

ID32

ID34

Preliminary calculation for LERSpectra of ID27A SX &HX beam lines

Global Ocean Sampling Expedition

• Sorcerer II of Craig Ventor Institute• GOS analysis identified some 6.12 million

proteins from 7.7 million newly discovered sequences

Human microbiomes• Within the body of a healthy adult,

microbial cells are estimated to outnumber human cells by a factor of ten to one.

• Human gut: 1000 microbiomes, 1x1014cells, 1 kg per person, but so far only 40 species have been characterized.

• Many sequencing projects in Asia, US, and Europe:– The NIH Roadmap Initiative now includes

a Human Microbiome Project (HMP,http://nihroadmap.nih.gov/hmp)

Acknowledgements Part 1Photon Factory, IMSS, KEKRyuichi Kato (talk on Wed PM)

Noriyuki Igarashi

Masahiko Hiraki

Masato Kawasaki

Naohiro Matsugaki

Yusuke Yamada

Leonard M. Chavas

Hisayoshi Makio (MKLP1/Arf6)

Norio Kudo

Kentaro Ihara

Tamami Uejima

Simin Rahighi (NEMO/Ub)

Seiji Okazaki

Kensuke Nakamura (Arl1/Arfaptin)

Ken Ohkubo

Accelerator Lab, KEKDivision VII Light Source

RIKEN M. Yamamoto

G. Ueno

K. Hirata

A. Nisawa

Y. Kawano

T. Hikima

H. Murakami

D. Maeda

T. Tanaka

H. Kitamura

JASRIStructure Biology G.

T. Kumasaka

N. Shimizu

K. Hasegawa

S. Baba

Light Source &Optics Div.

S. Goto

H. Ohashi

K. Takeshita

S. Takahashi

H. Yamazaki

T. Takeuchi

H. Yumoto

Control & Computer Div.

T. Ohata

Y. Furukawa

T. Matsushita

Acknowledgements II

Osaka UniversityAtsushi NakagawaMamoru Suzuki

Nagoya University

Nobuhisa Watanabe

Financial Supports

Protein 3000 Project (MEXT)

Development of Systems and Technology for Advanced Measurement and Analysis (JST)

Vesicle transport: Kazuhisa Nakayama, Kyoto Univ, Pharm. Dept

Hokkaido UniversityIsao Tanaka

Kyoto UniversityKunio Miki

Target Protein Research Program (MEXT)

Astellas Pharmaceutical Inc.

Thank you for your attention!Grand-in-Aid for Young Scientists (B) 18770098 (MEXT)

Linear Ub: Ivan Dikic & Fumiyo Ikeda, Goethe Univ, Frankfurt

Slide Number 1Slide Number 2Nobel Award in Chemistry, 2009Slide Number 4How large is the protein universe?Slide Number 6What do we want to know?Slide Number 8Protein-protein interaction networkSlide Number 10Transport in nueronsSlide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Time lapse imagesSlide Number 18Slide Number 197 lysines, K48, K63 and LinearSlide Number 21Slide Number 22Slide Number 23Slide Number 24Plethora of ubiquitin chains may lead to numerous biological signals ⇒ New ubiquitin world Slide Number 26Slide Number 27Slide Number 28Extremely large complex, Vault� (Tsukihara Group, Science 2009)�Vault crystal （Tsukihara et al., Science 2009, SPring-8 BL44XU）How many unit cells in micro/nano crystals do we need for structure determination?Micro crystals of cypovirus polyhedra�by Peter Metcalf et al.Laue function of micro crystalsTarget Protein Research Project (MEXT) �Microbeam Beamline BL32XU�Achieved beam size （2009/11/27） by SPRing-8Microfocus beamlines around the worldCrystal size and diffracting powerDevelopment of Micro-crystal handling �with laser tweezers being developed by SPring-8PF-AR NW14A: ERATO Project (~2009): Dynamics studies with 100 ps time resolution for innovation in materials and biological sciences�Tokyo Institute of Technology, ERATO (JST) S. Koshihara、KEK・PF　Shin-ichi AdachiAcetylcholinesterase�Prof. Joel Sussman et al.�Weizmann Institute, IsraelSingle molecule structure determination with hard x-ray FELDrug design targets: can we freeze the complexes?Ultimate storage ring and/or ERLSlide Number 43Preliminary Design of 5GeV ERLSlide Number 45Preliminary plan of�LER West-Symmetry Point Experimental hallPreliminary calculation for LER�Spectra of ID27A SX &HX beam linesGlobal Ocean Sampling ExpeditionHuman microbiomesAcknowledgements Part 1Acknowledgements II