icfa future light source workshop, 2 march 2010, stanford ......2010/03/02 · 2.5 gev pf science...
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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
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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
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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�
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The Worldwide Protein Data Bank (wwPDB) http://www.wwpdb.org/index.html
David S. Goodsell, Scripps Institute
>60,000 entries and growing!
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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
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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
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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?
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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?
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Protein-protein interaction network
Taken from Rua et al., Nature 2005
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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
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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
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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/
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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
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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
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微小管microtubules
MKLP1
Exocyst complex
Cyk4
Arf6 Arf6
細胞膜 Plasma membrane
分裂溝Cleavage furrow
+ -+-
+-
Rab11
FIP3Rab11
Fusion
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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
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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)
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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
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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
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7 lysines, K48, K63 and Linear
Lange et al.,
Science 2008
Taken from Dikic, Wakatsuki, Walter, Nat. Rev. Mol. Cell Biol., in press
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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
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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
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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!
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(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!!!
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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
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(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
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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)
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•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
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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
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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
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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)
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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
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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
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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
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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�
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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
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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
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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
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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�
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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
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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
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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
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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
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Preliminary Design of 5GeV ERL
XFEL-O
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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
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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
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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)
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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
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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
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