Cheiron School 2008 by AOFSRR SPring-8
5 October 2008

Protein
Crystallography
High Energy Accelerator Research Organization (KEK)
Institute of Materials Science
Photon Factory
Structural Biology Research Center

Soichi Wakatsuki

soichi.wakatsuki@kek.jp
http://pfweis.kek.jp/eng/index.html
1Outline
1. Introduction to protein crystallography
2. High throughput technologies for synchrotron
experiments
systems approach
3. Structural proteomics on post-translational
modification and transport of proteins: protein-protein
interactions in membrane traffic2Synchrotron Facilities with PX in Japan
Protein 3000 (FY2002-2006)

and new ones coming up

SPring-8
Saga
Synchrotron
1.4 GeV

Photon Factory
NagoyaSynchrotron
1.2 GeV3

Role of Structural Biology
GenomeDNA→RNA→

Proteins

Molecular machines（ribosomes,
enzymes）
Information network

ProteinProteintransport
transport

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

Biology(Basic research)

Medicine, drug design(industrial applications)4E. Coli cellDavid S. Goodsell, Scripps Institute, http://www.scripps.edu/mb/goodsell/5

Endocytosis of toxin by a clathrin vesicle

David S. Goodsell, Scripps Institute, http://www.scripps.edu/mb/goodsell/6

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

COLORS: proteins in blue, ribosomes inmagenta, DNA and RNA in red and orange,
lipids in yellow, and carbohydrates in green.
David S. Goodsell, Scripps Institute7

The Worldwide Protein Data Bank (wwPDB)

http://www.wwpdb.org/index.html

David S. Goodsell, Scripps Institute8

Acetylcholine esterase
Prof. Joel Sussmann
Weizmann Institute, Israel• ..¥Joel Sussman¥richardnew.mpg

9

Flow chart of protein
Future: automated/integrated system
crystallographic analysis
Crystallization robot

Crystal harvesting robot
Expression and purification

Crystallization

Crystal harvesting

Automated data collection
Data analysis

Mouting & data collection
10

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

Crystallization
robot（１）Large scale protein
expression and（３）High brilliance microfocus MAD beam line
purification system
Minipole Undulator

PF-2.5 GeV SR Ring

Automated
crystal exchange
robot andalignment system
inside the hutch

2D area detector（４）Automated protein crystal structure
analysis suite
11

ATG AAGGuanine nucleotide exchange factor Sec12p
1 atgaagttcg tgacagctag ttataacgtc gggtatcctg cgtacggtgc aaaatttttg
M K
61 aataacgaca cattacttgt ggcaggcggt ggaggagaag gaaacaatgg cataccaaac

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 atggtaaggt tgttgcttat atcaccggtt ctagcttgga agtgatttca

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
12

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 frommolecule to molecule are coloured the
same.
13

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
14

Protein Crystallization and crystal observation robot system
Observation system

Incubators

Dispensing system

Control PC
Carrying robot

15

Fig. 3
A

B

C

D

E

F

G

H

16

Protein Crystallization and crystal
observation robot system
Speed: 1 plate/36 sec = 2,500 plates/day = 240,000 trials/day
Sitting drop volume: 500 nL later down to 50 nL
Plate stacker
Incubators

Precipitant
pipeting

Protein pipeting
Sealing device
Observation
systemCrystallization plate

Plate handling Robot
17

Protein Crystallization and crystal observation robot system
・Fastest crystallization robot
Dispensing ：Sitting drop vapor diffusion: One plate (96 wells) every 36 seconds：180 seconds for each plate(from dispensing to sealing)
About 2,500 plates per day
About 240,000 drops per day
One batch capacity: 8 proteins, 960
precipitants,
80 plates

Example: 500 conditions:
2 days by manual operation→ 10 min (everything included)
・Web browser for crystal growth observation– To be developed for collaboration and as auser facility
M d l
d
d bl

18

Protein Crystals

19

~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:
17 % (w/v) PEG3350, 0.2 M KH2PO4
Buffer:0.1 M Tris-HCl (pH 7.5)
Temperature:
20 ºC
20

Packing of proteins in a crystal
30~70 % volume of protein crystals is solvent!

Cocrystal

Peptide-tagged
21

22

23

Blue Tongue Virus
Core Particle 1
P21212755 X 796 X 825 Å3
ESRF ID14/EH3
Detector: Imaging Plate
Crystal to1250
det distance mm

570 Å(20 pixels)

Wavelength

0.918Å

Osc. Angle

0.1 degExp. Time

100 sec

Pixel size

100 mmBeam size

100 mm

FWHM

181 mm24

Methods to Determine Phases
Heavy atom ＭＡＤ
Light atom ＳＡＤ(multiple anomalous dispersion)(Single anomalous dispersion)
Introduction of
anomalous
scatterers

Wild type
crystals

・Seleno-methionine (SeMet)
・Heavy atom soaking

λ１
λ2
λ3

X-raysλX-raysMulti wavelength anomalous dispersion

Anomalous signal from light atoms (eg: S)・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

・No need for preparing heavy atoms: highly
applicable for very difficult cases for which heavy
atom derivatives cannot be prepared・Light elements need low X-ray energy for higher
anomalous signal
・Weak anomalous signal necessitates extremely
highly accurate data, thus high redundancy

Many successes

Still not plenty

25

Beam Lines
SPring-8

Crystal sizes: 50～100μm

Ultra high brilliancemicro beam BL
(new)

50μm

BL44XU（SPring-8 / Osaka Univ）

Crystal sizes: 20～30μm

BL41XU（SPring-8）

50μm

Needs extremely
strong beams

BL-17A（PF）

Microcrystals: < 10μm

50μm

NW12A（PF-AR）
BL-5A（PF）

Heavy atomlabeling isdifficultLow energy microfocus BLPF26

Phase determination using MAD
Bijvoet pairs(Multiple Anomalous Dispersion)

Bijvoet Differences

MAD experimental maps:
Anomalous difference Patterson

E2
E1

E3
E4

Se K edge

ΔFE32 (at the peak)
Dispersive Patterson
FE42 - FE22 (remote – inflection point)27

Absorption edge measurement
X-ray
Iin

I0ut
IＦ

Iin
I0ut
I0ut

Energy28

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

SR Ring

2D area detector

1 degree oscillation29

30

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 Map31

32

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 heavy atom positions:
FH(+) & FH(-)
33

From intensity measurements
|FP|

Im

|FPH(-) ||FPH(+) |ϕP (?)Re

From Se positions-FH(+)|FP|-FH(-)34

Im|FPH(+) |-FH(+)ϕP-FH(-)Re|FP||FPH(-) |35

Met94

Met84
36

Ribbon diagram of trimer of GGA1 GAT domain
Triangle is threefold axis.
37

X-ray Area Detectors for Synchrotron X-ray Protein Crystallography
Tapered fiber optics CCD

Imaging Plates

Lens-coupled CCD

Flat Panel Detectors

HARP based Field
Emitter Array (FEA)

Pixel Array Detectors (PAD)

X-ray

38

X-ray Area Detectors for Synchrotron X-ray Protein Crystallographic Data Collection
Detectors

Pros

Cons

On-line imaging
plates

Large dynamic range, large area

Slow readout (20 to 200 sec per image) ->poor duty cycle
Relatively broad PSF
Relatively inexpensive

Off-line imaging
plates

Large dynamic range, large area

Slow read-out (20 to 200 sec per image) > poor duty cycle
Relatively broad PSF
Cumbersome to handle

Tiled, tapered fiber
optics CCD

Fast readout (0.3 to dozens of secs)

Limited dynamic range (~<16 bits)
Expensive to cover large solid angle

Lens-coupled CCD

Large active area (300 mm diameter)
Inexpensive

Has gone into market very recently, andnot yet established.
Large (1 m long) and heavy (100 kg)

Flat Panel
Detectors

Inexpensive, very light (~7 kg)
Large active area (easily 400 mm
square)Fast readout (a few seconds)

Inherent problem of noise

Pixel Array
Detectors (PAD)

Extremely good PSF
Extremely Fast readout

Still under development
Difficult to tile the components to cover alarge solid angle

HARP based Field
Emitter Array(FEA)

Extremely sensitive (800 X CCD)
Large area and very fast readout
Very good PSF

At the very first stage of the development

39

Detector Requirements for good
PX data collection• Large, fast, reliable and inexpensive• Must be an integrated systemdata acquisition and storage
data analysis
archiving• Easy to maintain40

Next generation detector: large area, highsensitivity, high speed, high resolution
SPring-8 CMOS based flat panel detector development
X-rays
Structured CsI crystals

Existing state-ofthe-art
technology

CMOS sensor

Direct crystal growth of thephosphor on CMOS sensor

Pixel（１） Improvement of sensitivity and spaitial resolution
using structured CsI crystals（２） Cost down by using existing state-of-the-art
technologies
Courtesy of Dr. Masaki Yamamoto 41

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

SR Ring

2D area detector

1 degree oscillation
42

Continuous rotation method:(Near FUTURE)(eg. 1 X 90 degs rotation)Datatransfer
Insertion device

SR Ring

2D area detector

Crystal rotation
43

Continuous rotation method:(Near FUTURE)(eg. single sweep of 90 deg
rotation)

Complete
Dataset!

Insertion device

SR Ring

Need much faster detector:
Pixel detector, HARP etc. +3D profile fitting

2D area detector

End of crystal rotation
44

AR-NW12 (N. Matsugaki et al.)Experimental Hutch
X-ray mirror

U#NW12
Front End

shuttercamera base

Optical Hutchmonochromator
35

ELV hall

30

25

20

15

10

5

liq. N2 cooling system

NW Experimental Halldata analysis(computer r oom)

detectors andequipments

Optical hutch

Experimental
hutch

45

PF-AR NW12: Highthroughput MAD beam line
Total data collection time for 180 frames: 10 to 30 min
Maximum sphere ofconfusion of the axis isabout 2 micron at sample
position
Fast X-ray shutter

PSIC
Slits ＆
attenuatorLong camera distance and large area size of the detector allow the datacollection of crystals with large cell dimensions

46

NW12: data collection from very small crystals.
Tool box makes manual crystal mounting easy.
Motorized telescope
allows centering crystals
automatically

Cryohead

Rotation Axis

SSD

Maximum sphere ofconfusion of the axis isabout 2 micron at sample
position

Beam stop etc. are all
motorized and installedin “tool-box”

47

Crystallization

KEK Photon Factory
200,000 trials/day

Tsukuba Express(45 min to Tokyo)

Li nac

Structural Biology
Research Center

6.5GeV PF-AR

2.5GeVPF ring

NW12A (2003)

BL-6A

BL-5A (2004)

BL-17A (2006)
48

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 Tong

Cryo Nozzle

Cassettes

Fancy Box

Compatible with SSRL（USA）, SRRC（Taiwan） &AS (Australia), CLS (Canada)
Compatibility with SPring-8 and European
standards being worked
49

SAM System Family
ALS
12.3.1
CLS 08B1-1(CMCF2)

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

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

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

SSRL
Automated
Mounting system
50Less time for crystal exchange
Original single tong
Data collectionModeswitchingUpper: Diffractometer
Lower: Exch robot

Centering
70~80 sec

Dismounting

Dead time

Cassette movements
Mounting Shortening of exp time

Double tongs＋ saving time for mode change over＋ improving time for crystal centering
Data collection

8 sec

Parallelmovements

Double tongs crystal exchange on PF BL-17A
Double tongs mechanisms

51

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

Another crystal

Beam Line:
Wavelength
Beam size :Exposure time:

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

100μm

Provided by Dr. Tadanobu Tanaka of Showa University

52

Towards lower energy, higher brillianceBeam size at sample[μｍ]

Current operational
PF beamlines

２００

NW12A, BL-5A
BL-6A

１００

・After collimator
・Flux > 1010

Target of the Next PF Beamline BL1A
BL-17A(today)０５

Use of lower energy

１０Higher brilliance
１５

２０ [keV]

Energy53

BL17: Mini-gap undulator &Experimental environment• High speed shutter
Precision< 1 msec.

• High precision goniometer-head
SOC (rotation error)< 0.7 μm

• Fast readout, large area and highgain CCD detector (ADSC Q270)
Readout
1.1 sec
Active area 270 x 270 mm2
Gain
21 e-/photon at 12.4 keV
Minimum distance 40 mm• Automatic sample changer
Exchange< 11 sec.

54

BL17 example
Se-MAD experiment: data collection at three xtal
positions to avoid radiation damage
University
Atsushi Nakagawa, Harumi• Osaka
DNA binding
protein
Hosaka• Mol weight：37 kDa•

Xtal：2 x 10 x 150 μm3•

3.2 Å resolution （Se-SAD）•

Data collected at 3 positions

30μm

After density modification
55

High precision one axis diffractometers with XYZ stages
BL

BL-6A

NW12

BL-5

BL-17

Year started

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)0.6

変位量 [μm]

0.40.36μm0.2
00.6 μm

-0.2
-0.4
-0.6
0

Air bearing & magnetic x-y stages

100

200

300

ω角度 [degree]

BL-5 type diffractometer -> also installed on BL41XU & BL44XU, SPring856

SPring-8 BL41XU experimental hutch
Cryostream (He gas)

Cryostream (N2 gas)
Vacuum Chamber*Back Light*Beam Stopper
Cameras*He-flowed Collimator
Goniometer*Detector (Q315, ADSC)

Experimental Stage (RIGAKU)
57

Industrial Use and Collaborations
between KEK and Industry (~8% of beamtime)

MX BLs at Photon Factory

Ajinomoto Co., Inc
Astellas Pharma Inc.

Astellas Pharma.
Beam Line ARNE3 (to becompleted byMarch 2009)
Company ①
Company ②
Company ③

Tsukuba Structural
Biology Consortiumfor 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
58Astellas Pharma Beam Line: PF-AR NE3

Available from Apr. 2009

NE3 Undulator

Optics Hutch
CM DCM FM

FPExp. Hutch• Expected to become stronger than AR-NW12A• Astellas Pharma will have priority 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.

59

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

60

Protein glycosylation and transport
Endocytic pathway

Protein glycosylation

GGA

Autophagic pathway

T. Yoshimori

61

Protein 3000 (Ministry of Education, Culture, Sports, Science and Technology)
Network Committee forProtein Analyses
500～600 structures, 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.
Inst. for Protein
Research

Other ResearchCenters of RIKEN
Beam time use

Collaboration

Collaboration
Collaboration

Beam time use

Signal Transduction
SPring-8
Higher Order Biological
FunctionsBrain and Neurology

Corporation
in R&D and
facility
operation

Beam time use

Industry

Photon Factory
Beam time use

Metabolism
Collaboration

62

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

KEK, Photon Factory (PX, SAXS)
Nagaoka Tech Univ (X-ray)

Nagoya City Univ. (NMR)
Tokyo Inst. Tech (PX)

Nara Inst. Sci. & Tech (SAXS)JAERI, Nara (Bioinfo)

Original goal: 70 in 5 years (2003-March 2007)⇒ changed to 150 last year
254 structures solved (31 March 2007)
Osaka University
Inst. Microbial Diseases
Kyoto University
Pharmacology Department
NAIST,
Molecular Biology Lab
Kyoto University
Life Science Program

171 PDB deposits

Kagoshima University
Medical School

University of Tokyo (Komaba)

Riken, Membrane
Traffic Group

Osaka University
Medical School
Tokyo Metropolitan
Institute of Gerontology

Riken
Immunology InstituteKyoto Sangyo University
Faculty of Engineering
63Collaboration outside of Japan
1. Human sialidase: G. Tattamanti & G. Monti, Italy,(Chavas et al., JBC, 2005)
2. Human sialidase inhibitors: M.v. Itzstein, Institutefor Glycomics, Griffith University, Australia
3. Sialidase inhibitors: Peter Colman, Australia
Steve Withers, Canada
4. Endocytic pathways: H. Stenmark, Oslo, Norway(Slagsvold et al. JBC, 2005, Hirano et al. NSMB,
13, 272, 2006, Hirano et al. NSMB, 13:1031, 2006)
5. Protein carbohydrate recognition in HIV infection:
R. Varadarajan, Bangalore, India
6. Protein carbohydrate interaction, Johan Deisenhofer,
Univ. Texas, USA (C.-I. Chang et al. PNAS, June 2005)
7. Ubiquitin recognition, Ivan Dikic, Johan Wolfgan Goethe
Univ.
Frankfurt, Germany
64Model for intracellular transport of N-linked glycoproteins65Complex structure of the Ca2+/Man2-bound VIP36
exoplasmic/lumenal domain

Stalk domain

Tadashi Satoh et al., J. Biol. Chem. (2007) 282, 2824666

Model for binding between VIP36 and high-mannose typeglycoprotein, salivary α-amylase

Tadashi Satoh et al., J. Biol. Chem. (2007) 282, 2824667

Small GTPases involved in
vesicle transport:
Rab and Arf

68

Small GTPases, ARF and Rab, in vesiculartraffickingT. Shiba, et al., Nature Structural Biology 10 386-393, 2003

T. Shiba et al. PNAS 103, 15416, 2006
69

Taken from I. Jordens, et al., J.Neefjes, Traffic 2005; 6: 1070–1077 70

Transport in nuerons
From N. Hirokawa, J. of Neuroscience, 2006

DoubleheadedkinesinOgawa, et al., Cell, 2004

KIF1A: single headed motor, Nitta, Hirokawa et al., Science
2004, 305, 678 – 683, Data collected on PF-BL6A & 19ID APS
71

Membrane recruitment of effector
proteins by Arf and Rab GTPases

FROM: Munro S.
Organelle identity and the targeting of peripheral membrane proteins.
Curr Opin Cell Biol. 2002, 14: 506-514.

72

Small GTPases involved in
vesicle transport:
Rab and Arf in GTP form:
effector interactions73

Endocytosis of toxin

David S. Goodsell, Scripps Institute, http://www.scripps.edu/mb/goodsell/74

Clathrin
movie byAllison
Bruce,
HarvardM. Marsh and H. T. McMahon, Science, 1999, Vol.
University
285, 215
http://www.hms.harvard.edu/news/clathrin/
75

Human GGA: a new class of adaptor proteins
GAT
Clathrin
terminal domain① VHS

Clathrin

GAE③

ARF-GTP
M6PR

②
MPR

N-GAT

Autoinhibition

Accessory protein

clathrin box
Hinge region

Competition
TGN membranewith AP-1

Lumen① Shiba et al. Nature 415,937-941, 2002
cargo protein
Shiba et al., Traffic, vol. 5, 437-448, 2004
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

76

GGA-GAT is indispensable for docking onto the TGN membrane
GGA-GAT
C
303

235
299

206

C

245

ARF1-GTP

166
N

α1’

α3
166

171
α0

α2

275

184

α1

186

269

C

N

199
N

Trans Golgi Network membrane

192

GAT domain apo-form

N-GAT & ARF1-GTP complex
77

Interaction surfaces of N-GAT and ARF1-GTP
GGA-GAT docks on the membrane via mostly hydrophobic
interactions with Switches 1 & 2 and interswitch region of ARF1-GTP
Interswitch

N

N-GAT

ARF1

C

L190

Switch 2

L182
N194
K198

A193
L178
I197

V201

C

F169
N

Switch 1

W66
Y81

A179
H80

F51
G50
L77

I49

K73
The residues involved in theinteractions are indicated byball-and-stick models.

78

Arf’s interacation site is used by effectors and many other proteins(Kawasaki, Nakayama, &Wakatsuki, Current Opinion in Structural
Biology 2005, 15, 681)
Exploitation of the human protein transport pathway by cholera
toxin.
Arf1–N-GAT

Arf6–CTA1（Cholera toxin）

N-GAT
GGA1

VHS

C-GAT
1

147 166 210

CTA1

GAE
305

510

C

639

1

192

C

N
N

I74
I49
G50 I197 L77
Y81
F51
V53

W66

C

I70
V45
G46 N93 L73
Y77
F47
V49

W62

N

T. Shiba, et al., Nature Structural
Biology 10 386-393, 2003

Structure by Wim Hol, Seattle, USA
C.J. ONeal, et al. and W.G.J. Hol, Science 12
August 2005
79

FIP (Family of Rab11 interacting
proteins)

Pull down
assay using
HeLa cells

FIP3/4 can also interact with
ARF5 and Rab11 simulateously.
80Rab11/ Rab11-binding domain (RBD) of FIP3
T. Shiba et al.
PNAS 103:
15416, 2006.
Also:
FIP3/Rab11 byLambright,
JMB, Aug 26,
2006&
FIP2/Rab11 by
A.R. Khan,
Structure, Aug
2006
Target membrane81

Two-fold symmetry of Rab binding domains
Rab5–Rabaptin5N

Rab7–RILP

Rab11–FIP3

N

N

N

N N

C

C

N

C

C
N

C
C

C

C
C

C
N

N
NC

CN

Membrane

Membrane

Membrane

Zhu G, et al., Nat.
Struct. Mol. Biol.
11, 975-983 (2004)

Wu M, et al., EMBO
J. 24, 1491-1501(2005)

Shiba T. et al.
PNAS 103, 1541615421 (2006)82

Two-fold symmetry of Arf/Rab binding domains

N

N

N

C

N

C

C

N

Rab6–GCC185

Arl1–GRIP
C

N
C

C

Membrane

Burguete AS, Fenn TD, Brunger AT, Pfeffer
SR, Cell 132, 286-298, 200883

Studies towards Structure Based Drug Design
Anti-influenza drug with little or no side effects
Neuraminidase (N)Oligosaccharides
with sialic acids
Hemagglutinin (H)Entering

Budding

Influenza type: H5N1

There are 4 sialidases in human which
resemble influenza neuraminidase:drugs against influenza neuraminidases
might affect the human sialidases.
84

First human sialidase (~neuraminidase) structure
Neu2 folding and electrostatic surfaces

Ribbon diagram representation(view from the active site)

Electrostatic surface representation(blue and red regions are basic andacid surfaces respectively)

Chavas et al., J. Biol. Chem. 2005 vol 280, 469-75
85

Zanamivir bound in humansialidase Neu2
Y334

R304

R237
R21

L217
I22

E39

M85
Q111

Y181
86

Structural Differences between Human Neu2 andInfluenza Virus Neuraminidase
R374

R304

Y409
Y334
R21

⇒ Towards
developments of new
Influenza virus
inhibitors withoutside
R237effects
R292

R116
T275

Talk by Leo
Chavas, 23rd
Session 7, 16:35

D46

Y179

D149
R223
R150

E111

Y181

Leo Chavas et al., J. Biol. Chem. 2005
Human Neu2
vol 280, 469-75
Influenza virus neuraminidase (1VCJ)87

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

Lead compounds

Tamiflu
derivedZanamivir。
Inhibitor design

Clinical tests
Selection

Active site

Structure Determination using SR
Drugs with noside effects:
Interaction with
only influenza
neuraminidase
but NOT withhuman sialidases

Tamiflu
Difference

Yellow: human sialidase Neu2
Gray: Influenza neuraminidase

Human sialidase
Neu2 structure

Side effects:
Humansialidases?
Neu1, Neu2,
Neu3, Neu488

Target Protein Project (5 years: 2007-2011)• Yearly budget: 5.5 billion yen, US$ 44.5M, 33.3 MEuro,(includes 30% overhead)• Proposal deadline: 20 April 2007
• Selection results published: 15 June 2007
• 43 teams selected
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
89

Target Protein Project (2007-2011)
The Analysis Core
Joint Proposal by SPring-8 and PF: Two New Beam LinesPF② Microfocus
Beam Line
Low to medium
energy (Invacuum
short gap
undulator)
1.
2.
3.

① Microfocus
Beam Line
Medium to highenergySPring-8

To be completed by 2009
Compatibility: SPring-8 and PF robots
Remote access and control

Isao Tanaka, Hokkaido Univ
Kunio Miki, Kyoto Univ
Atsushi Nakagawa, Osaka Univ.

90Beam Lines
SPring-8

Crystal sizes: 50～100μm

Ultra high brilliancemicro beam BL
(new)

50μm

BL44XU（SPring-8 / Osaka Univ）

Crystal sizes: 20～30μm

BL41XU（SPring-8）

50μm

Needs extremely
strong beams

BL-17A（PF）

Microcrystals: < 10μm

50μm

NW12A（PF-AR）
BL-5A（PF）

Heavy atomlabeling isdifficultLow energy microfocus BLPF

91

Target Protein Research Project (National Project)
SPring-8

PF

Micro-focusing beamline for tiny
crystals (<10 μm)

Micro-focusing beamline for sulfurSAD phasing

Goal：
1010 photons/sec/μm2@ 12 keV

Goal：
1010 photons/sec/10x10μm2@ 4～5 keV

Current: 1012 photons/sec/100x100μm2 (BL41XU)
4

Current proposalf"(Electrons)

3.5
3
2.5
2
1.5
1
0.5

P
S
Se

BL17
A

0
4

Beam profile of SPring-8 BL41XU

5

6

7

8

9

10

11

12

13

14

Energy (keV)

Further increasement of flux

User operation from April 2010.

92

Beamline design of BL32XU @ SPring-8
by M. Yamamoto et al.

Concepts
1. Making the best use of capability of SPring-8 undulator
2. Minimizing a number of optical elements to reducemechanical errors
3. Focusing with a LARGE magnification factor
93Current beamline control system @ SPring-8
by M. Yamamoto et al.

Multiple X-ray exposures on one/several crystals to
avoid serious RD during data collection
・Vector Centering：・Multiple Centering：

Changing exposure position linearly inevery constant frames on One crystal

Multiple exposure positions using
several crystals in One cryo-loop

25μm

①
②

③
⑥
④
⑤

25μm

94

Current status of construction of BL32XU @ SPring-8
Design & ray trace
Frontend
Beamsize 1 x 2 mm2Stage for elements

Flux density

1016 phs/sec/mm2

High-precision DCM

X-ray shield hutch

R & D of CCD detector

High rigidity
High precision95

X-ray shield hutch of BL32XU @ SPring-8

Start
2007/12/21

Before construction96

X-ray shield hutch of BL32XU @ SPring-8
Finished product...

2007/12/21 ~ 2008/02/25

SophisticatedJAPANESE workers
97In-vacuum Short Gap Undulator Microfocus
Beam Lines: softer X-rays @ Photon Factory
NEW BL1A

4
P

NEW BL1A

3.5

S
Cl

3
f"(Electrons)

K

2.5

Ca

BL17A

2

Se

BL17A

1.5
1
0.5
0
4

5

6

7

8
9
10 11
Energy (keV)

12

13

14
98

Front end

Lower Energy
Microfocus undulator
beam line PF-BL1A

Liq N2 refrigeration unit
for DCM
Vacuum components

Wire BPM

KEK-PF BL-1A: in-vacuum short gap undulator: fundamental to cover 4.1~4.5 keV99

Towards lower backgroundby Isao Tanaka, Hokkaido University
Loopless crystal mounting method

New scheme

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

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

100

Towards lower background:
Promising techniques developed by other groups.
OR CUT AWAY the rest with a 193 nm UV laser

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

101

102

Micromanipulator
systemIn collaboration with Dr.Tanikawa (AIST)103

Crystal handling: so many different systems…

BAM

SPACE

SBC-CAT

mar-CSC

EMBL-Hamburg

ACTOR

EMBL-SC3

PLS

DORIS

BruNo

CATS

SAM

Cool-Hand-Luke
104Compatibility with other systems

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,…
105

Summary• Target Oriented Structural Proteomics of Protein
Transport• New insertion device beamline, BL-17A, has beendeveloped at the Photon Factory 2.5 GeV ring.
• Recent progress on the stabilization of X-ray beams
and the development of the extremely highprecision diffractometer enable more precise datacollection.
• New Target Protein Research Program: challenging
protein targets (membrane proteins and complexs):
two complementary micro beam BLs at SPring-8
and Photon Facotry• New microfocus lower energy PF-BL1A will be used
fo lower energy SAD phasing (and normal MAD).
• Various exchange mechanisms for cryo-pins andexchange robots
106

Acknowledgements
SPring-8
Masaki Yamamoto’s group
Hokkaido University
Isao Tanaka
Kyoto University
Kunio Miki
Osaka University
Atsushi Nakagawa
Mamoru Suzuki

Financial Supports
Development 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
107Structural Biology Research Center, Photon Factory, KEK
Soichi Wakatsuki

Professor, Director of Structural Biology Research Center

Structural Biology
Ryuchi Kato (Associate Prof.)
Masato Kawasaki (Research Assoc.）
Kentaro Ihara （Post Doc Fellow）
Norio Kudo （Post Doc Fellow）
Mohammad Yousef (JSPS fellow)
Tamami Uejima（Researcher）
Tamie Aoki（Lab Technician）
Honda （Lab Technician）
Ex members:
Tadashi Satoh （Post Doc Fellow）
Leonard Chavas (Post Doc Fellow)+++

Beamline & Robotics
Noriyuki Igarashi （Research Assoc.）
BL-6A, BL-17
Naohiro Matsugaki （Res. Assoc.）
AR-NW12, BL1
Masashiko Hiraki （Res. Assoc.）
Robotics
Yusuke Yamada （Research Assoc.）
NE3
Yurii Gaponov （Researcher） Programming
Kumiko Sasajima（System Engineer）
Shokei Watanabe （Mechanical Engineer）
Nobuo Honda（System Engineer）
Kazutaka Demura（Beamline Assistant）
Kubota （Beamline Assistant）

The Graduate University for Advanced Studies
Junichi Kouyama (D4), Simin Rahigi (D2), Ahmed Rohaim(D1), Kensuke Nakamura (M1), Ken Ohkubo (M1)

Secretaries
Naomi Nagata*
Tomoko Zeniya*
Ritsuko Ebisawa*
Reiko Sukegawa

Internship Students: Atsuko Tsuchiya (Kyoto Univ. M1), Ryunosuke Nakatani (Nagaoka Univ. of Tech. B4)108