Towards the femtosecond crystallography using SACLA on
membrane protein crystals

So Iwata
Kyoto University/Riken SPring-8 Center (SACLA)

Membrane Protein Crystallography Pipeline based on
Recombinant Expression Systems Focusing on
Mammalian Targets• Method development to address major bottlenecks for membrane protein
crystallography.
Keyparameters tomonitor

Processes• Activity• Expression level• Integrity• Indexing• Scaling andconcentration• Resolution

merging

Expression

Purification

Crystallisation

Data collection~500~200~60~30•Constructdesign•Expression
options/developments systems
Technical• Monodisper-sity• Thermal stability• Detergent• Cultureconditions• Detergentchoice• Purification
methods• Additives• Crystallisation

methods• Detergent choice• Additives•Antibody

fragments

Structure
determination
10• Data collectionsystems• Samplemounting•XFEL• Phasing methods• Model building
and refinement

!"%#Membrane transporters & respiratory enzymes(")("&+*%"

$!#)

,&'

"

Mhp1 hydantoin transporter(Science 2008, Science 2010)

In
Oligopeptide
transporter (EMBO J.
2010)
B.

ASBT bile acidtransporter (NatureCore in press)
Pa2011,#$+&,(-!

OutInC.

NO reductase- Fab complex(Science 2010)C

Human anion exchanger - Fab complex

CorePanel!

CoreG-protein coupled receptors
Human Adenosine A2aR-Fabcomplex
IV(Nature2012)
III

Human Histamine H1Ra(Nature 2011)
ECL3

ECL2

S-Sb
ECL1

ECL1
ECL2

NS-S

PO43Doxepin

ECL3

D107
W428

VII II

V VI
IVIc

NPxxY

V

VIII
ICL2

II
Antagonist
Doxepin(ZM241385)
VI

D(E)RY

ICL3

VIII

V

III

CWxP

Human Muscarinic acetylcholine
M2R(2012)CVIII
H 1R N!2ARIV

A2a IIIECL1
ECL2
receptor
VIIIDoxepin

ECL3 i3-loopVI VII

ICL1CCIII
N
H 1R
D3R

Fig.1

AntibodyFabFragment

Antihistamine (Doxepin)

CDR-H3Inverseagonist

How fast can we solve a membrane protein
structure?

Stabileconstructdesignex. )Histamine
H1

7 months（2009/3−2009/11)

3 months

Achievablespeed in 5years

Crystallization

7 months（2009/11−2010/05)

3 months

X
5< 1 month

Datacollection/ analysis
7 months（2010/5−2010/1)

Compoundscreening

1 month（2011/7−2011/8)

GFP-based Membrane Protein Expression andPurification System in S. cerevisiae (1)MP-TEV-GFP-8His

1.Screen membraneprotein-GFP overexpression by wholecell fluo-rescence

TEV 10His

NGFP

Drew et. al. Nat.
Methods. 2, 2006
Newstead et. al. PNAS
104, 2007
Drew et al., Nat.
Protoc. 3, 2008

8His
C

2. Isolate crude
membranes

3.Check integrity
using in-gel
fluorescence

1.Detergentsolubilization

2.Incubation ofsupernatant with NiNTA resin

3 . I M A C
purification

Ni-NTA resin

Spin downnondetergentsolubilized proteins

4. Check cellular
localization by
confocal micro-scopy

5.Check monodisperisty in different
deter-gentbyfluorescence sizee x c l u s i o nchromatography

Flow-through4.Dialysis andcleavage of
GFP-8His

5.Reverse IMAC toremove GFP-8His
and his-tagged
TEVConcentrationof
membrane protein

WashElution

6.Gel filtration of
membrane protein(option)GFP-based Membrane Protein Expression andPurification System in S. cerevisiae (2)• Suitable for fast screening of the most stable/expressed constructs

S. cerevisiae

onatim,rion
R foectl
PCranse-sT
Ura

11~13 daysnele
Vectoresatio lectionnimcri
constructionoenetnsf MD sGeTractio

nP. pastoris

19~23 days
Vectorconstruction

Bacmid.

Insect cells

Traionectfsn
P1 virus

105P2 virus

20

1530~40 days

Titer check

25

30

35

Easy to transfer tothe systems to
produce better
proteins

Days

Sugawara et. al.
BBRC. 704, 2009

• Easy to construct variants by combining multiple fragments
Primer 1 & 2
Primer 3 & 4
Primer 5 & 6+pDDGFP-2 vector

1-29

N5Q, N18Q

F116W

" !#"" !

$ "              

  

472-48720222

T4L
HRH1$Nd$T4L

404487i3d (229-397)

Crystallisation of Membrane Proteins usingDetergents• Large micelle detergents stabilise membrane proteins• Small micelle detergents are more suitable for crystallisation• Antibody fragments facilitate membrane protein crystallisation even inlarge micelle detergents

Antibody Fv
Cytochrome c fragment
oxidaseMembraneprotein

Using smaller
detergent

Membrane protein crystals
Antibody Fab orFv fragment

Detergent
micelleExtend polar surface
using antibody fragment

Crystals of themembrane protein antibody complex

Cytochome c oxidase Antibody Fv fragment
crystal (Iwata et al.
Nature 376, 1995).
8

New structures (1)

1.9Å

Human
erythrocyte
anionexchanger 1(Band 3) - Fabcomplex

Mouse sugartransporter Fv complex
Diffraction imageat SACLA

New structures (2)Supplementary Figure a.
1

CoreDimer

MmRce1
N

1a.

1

2

77
Fab (H)

9

177

Fab (L)893 a1164b811a6101212b.

Dimer

Core2

111b

194b124a11a11b8

2
94b693Core1011b257Core

1Dimer12101C1 Core
12Dimer10b.
Dimer

34bC90° 

11b 54a12  CoreDimerN 

11b 54a14a11a4b10 751238264a11a

NapA (outward-facing)

911b4bNhaA (inward-facing)+ antiporter
Supplementary
Figure 1 Structureof the complex
MmRce1-Fab645-2 complex. Overall ribbon
Membraneprotease
FabNapA
sodium/H
55
diagram of the MmRce1-Fab645-2 complex, with MmRce1 viewed7 parallel to the7(Nature in press)

membrane. MmRce1 is depicted in blue, the antibody Fab fragment heavy chain [Fab(H)] in yellow and the antibody Fab fragment light chain [Fab(L)] in green. MmRce1
interacts with Fab645-2 via its cytoplasmic side.

83(Nature in press)

8104a11a3

創薬ターゲット蛋白質の迅速構造解 SACLAを用いた超迅速構造解析 析法の開発

SACLA @SPring-8
Pulse Energy*
Peak Power*
Pulse duration
Photon energy range

0.3 mJ @10 keV>30 GW~10 fs or less
4.5 to 15 keV

Stability Intensity σδI/I *< 10%Repetition rate

20 Hz (Max. 60 Hz)

26/6/13&製薬協

Summary'of'Performance
Pulse Energy*
Peak Power*

0.3 mJ @10 keV>30 GW(1 billion times stronger than synchrotron)
Pulse duration~10 fs or less
(Shorter than chemical bond breakage time )Photon energy range
4.5 to 15 keV(Hard X-ray suitable for X-ray crystallography)
Stability Intensity σδI/I *< 10%
Repetition rate

(Very stable)
10 Hz (Max. 60 Hz)(Fast)* depending on the lasing wavelength12Serial Femtosecond Crystallography at SACLA

SACLA%design%
8*GeV
beamdump*

Thermionic*electron*
gun50*MeV
beamdump*

CBband*linearaccelerator*
SBband*linear
accelerator*

Short*period
inBvacuum
undulator

Bunch*
Bunch*
Bunch*
compressor*1 compressor*2 compressor*2Kicker*magnet*
Low*emi1ance
injector

Beam*monitor**

700$m

Experimental*
hutch~700$m

ICSG2013,SLS&13

Protein nanocrystallography using X-FEL• Photosystem I structure has been solved at LCLS(Stanford,US) using nano crystals ( 200 nm to 2 µm in size) at 8.5 Åresolution (Chapman et al., Nature 2010).
• Using liquid jet system, 112,725 images were collected
RESEARCH LETTER(15,445 were used) .
• 70 fs pulses cause little radiation damage. Sample dose is ca
10700 MGy (cf. 30MGy limit for conventional synchrotron 10experiments) and this can be significantly improved with 10shorter puls.
10Th(1 mobt
Dint
2,4Intensity (photons)

410 fs
70 fs
200 fs32

1Fulthe100
10–1

0.1

0.2

0.3

60

30

200.4
150.5
120.6
10q (Å–1)d (Å)

Figure 4 | Pulse-duration dependence of diffraction intensities. Plot of theintegrated Bragg intensities of photosystem I nanocrystal diffraction as afunction of photon momentum transfer, q 5 (4p/l)sin(h) 5 2p/d (wavelength,l; scattering angle, 2h; resolution, d) for pulse durations of 10, 70 and 200 fs.
Averages were obtained by isolating Bragg spots from 97,883, 805,311 and
66,063 patterns, respectively, normalized to pulse fluence. The error in eachplot is indicated by the thickness of the line. The decrease in irradiance for
200-fs pulses and d , 25 Å indicates radiation damage for these long pulses,
which is not apparent for 70-fs pulses and shorter.
6down to a single unit cell (that is, a single molecule). As this limit isapproached, the ordering of the nanocrystals will become increasingly
irrelevant, as each crystal may be treated as a single object and the‘disorder’ that conventionally leads to reduced resolution will simply
manifest itself as shot-to-shot variability,14
providing information about
not just the average structure but also the range of dynamically accessible
conformations.
Data are collected on fully hydrated nanocrystals without cryogenic

Rec
1.
2.
3.
4.
5.
6.
7.
8.
9.

10.

11.

Serial Femtosecond Crystallography at SACLA

Data%collec2on%system%
Experimental#hutch
Data#collec+on#system
Injector#

Sample#loading#system

Opera+on#desk#

Detector##Sample#prepara+on#room

Valves#Opera+on##
system##DiﬀracOon*
chamberOﬄine#test#bench#oﬄine#injector#

Pumps#
Sample#shaker

Oﬄine&test&bench• To*facilitate*injector*(or*any*sample*loader)*development,*an*oﬄine*injector*testBbench*has*been*setup*next*to*the*sample*
preparaOon*room.*
Data&collec0on&system• A*sample*loading*system,*an*injector*and*an*mpCCD*detector*have*been*integrated*with*a*diﬀracOon*chamber*to*construct*the*data*collecOon*system*for*the*SFX*experiments.*• We*have*tested*the*system*using*Lysozyme*as*a*test*sample*(March*2013)*to*evaluate*the*system.*

ICSG2013,SLS&

Serial Femtosecond Crystallography at SACLA

Serial%Femtosecond%Crystallography%(SFX)%data%collec2on%
system%at%SACLA
Injector#Detector#DiﬀracOon*
chamber

SWDQmpCCD
Image#format:#2048#x#2048#(110mm#x#110mm)
SampleQdetector#distance:#min.#50mm
Nam#&#Song,#et#al.,#Imaging#fully#hydrated#whole#cells#by#..##(Phys.#Rev.#Le<.#110:098103,#2013);
Park#&#Song,#et#al.,#Assessment#of#radia>on#damage#in#single#..#(Phys.#Rev.#E.#86:042901,#2012). FrameQrate:#max.#60#Hz
Quantum#eﬃciency:#
80%@6#keV,#20%@12#keV#ICSG2013,SLS&

Serial Femtosecond Crystallography at SACLA

Liquid jet injector for SFXFeatures• X-ray diffraction from proteins in buffer solution (native)• Quick adjustment of liquid beam diameter (3~30µm): adaptable to crystals in various sizes• Lower sample clogging at the nozzle
Typical liquid flow rate (sample consumption):• 20µL/min for the 3µm liquid beam: ~ mL/hour• 0.2mL/min for the 30µm liquid beam: ~ 10mL/hour
SampleflowHe-gas
focusing

30µm

ICSG2013,SLS&Plan%for%FY%2014:%advanced%SFX
Advanced injectors under development
Recirculate samples:
Save sample consumption(requires down to a few ml ofsamples)- Recirculate the sample
reservoir

Synchronised#droplet#injector
Save sample consumption
significantly (1/10,000 of thecurrent injector or a few µg ofsamples)- Easy to control the sample
environment

Hybrid#ﬁxed#target:
Q#For#lipidic#cubic#phase#Q#30~60#Hz#raster#scanProtein nanocrystallography at SACLA（１）• Data collection system with a liquid jet and a fast CCD(mpCCD) has been constructed (March 2013)• 10 fs pulses cause almost no radiation damage because it isfaster than chemical bond breakage.
• More than 200,000 images can be collected in one hour at
60Hz (currently 36,000 images at 10Hz). It means we canfinish data collection within 30min or less.
• Data from 5-10 µm lysozyme crystals has been collected at1.5 Å resolution.

19

Protein nanocrystallography at SACLA（２）Only one in every 10 images are shownSerial Femtosecond Crystallography at SACLAFirst SFX data at SACLA（Mar 25th, 2013）~1.7#Å

Diﬀrac+on#pa`ern#from#a##
Lysozyme#crystal#using#liquidQjet#
injector#

ICSG2013,SLS&

Serial Femtosecond Crystallography at SACLA

Data processing
Fast#selec+on#

PickQup#the#images#which#have#pixels#more#than#
10,000#counts#within#the#water#ring#by#a#Python#
script#(6148images#）

Indexing#and#
integra+on
Program#:#Indexamajig
3520#images#were#indexed#using##CrystFEL##ver.#0.5.0#(Thomas#A.#White##et#al.)7#runs#(14,000#images)
12min#at#10#Hz#
Sta+s+cs#

Merging#

Program:#check_hkl

Program:#process_hkl

Evaluate#completeness,#
S/N,#redundancy#and#so#on.

Calculate#mean#intensi+es#of#the#diﬀrac+ons#with#the#same#index.#

ICSG2013,SLS&

Serial Femtosecond Crystallography at SACLA
Data processing of Lysozyme data
Run94362+,94368,7datasets,（14,000,images）↓6148 were selected （over 10,000,count,pixels）↓3520 were indexed then processed

1/d#centre#####refs###Possible#####Compl#######Meas######Red######S/N#######d(A)#####1.584#######2068#######2068#######100.00######97604#####47.2#####7.88###6.31#####3.438#######1943#######1943#######100.00######76769#####39.5#####7.94####2.9#####4.111#######1906#######1906#######100.00######69701#####36.6#####6.91####2.43#####4.609#######1885#######1886#########99.95######60245#####32.0#####5.54####2.17#####5.016#######1871#######1871#######100.00######49939#####26.7#####4.48####1.99#####5.366#######1864#######1866#########99.89######33923#####18.2#####3.73#####1.86#####5.675#######1845#######1857#########99.35######17821#######9.7#####2.97#####1.76#####5.953#######1792#######1867#########95.98######10594#######5.9#####3.11#####1.68#####6.207#######1361#######1847#########73.69########5052#######3.7#####4.26#####1.61#####6.442#########411#######1852#########22.19########1055#######2.6#####6.64#####1.55

Electron density
map（2Fo)Fc,$1σ）
20,cycle,reﬁnement,using,refmac,with,the,TLS.,Without,solvent,molecules.,%Res%%=%30+1.7%Å%(1.744+1.700%Å)!Compl%=%99.8%%(98.8%)!Rwork%=%21.3%%(44.1%)!Rfree%%=%25.3%%(53.9%)
Mean%B%=%29.9%Å2

ICSG2013,SLS&

Serial Femtosecond Crystallography at SACLA

Diﬀrac+on#pa`er#from#various#protein#samples#(including#those#structures#are#unknown)#（Ni1a*et*al,*Tokyo*Univ.)（Chavas*et*al、KEK)（Masuda*et*al.,*Kyoto*Univ.)（Chavas*et*al、KEK)
ICSG2013,SLS&（Kato*et*al.,*Kyoto*Univ.)（Mizohata*et*al、Osaka*Univ.)
24

Serial Femtosecond Crystallography at SACLA

Improved X-ray Diffraction of Mammalian Sugar
Transporter-Fv Complex Crystal at SACLA
Q#SugarQtransporter#Q#Fab#complex#crystals#
diﬀracted#up#to#3.2Å#at#BL32XU#of#SPringQ8.
QDiﬀrac+on#limit#was#signiﬁcantly#improved#
when#a#large#crystals#was#used#at#SACLA.#

SPringQ8

SACLA1.9Å

1.9Å

3.2Å

ICSG2013,SLS&Diamond-MPL(Supported byWellcome trust andBBSRC)David Drew
Alex Cameron
Simon Newstead

Hidden gems

Kyoto University (supported by JST
Research acceleration program)
Satoshi Ogasawara, Norimich Nomura,Takuya Kobayashi, Takeshi Murata, Tatsuro
Shimamura, Tomoya Hino, Takatoshi
Arakawa

Riken SPring-8 Centre (supported by X-ray
Free Electron Laser Priority Strategy
Program (MEXT))
Eriko Nango, Rie Tanaka, Jaehyun Park,Changyong Song, Takaki Hatsui, ErikoNango, Rie Tanaka (RIKEN)
Kensuke Tono,Yasumasa Joti, Takashi
Kameshima (JASRI),
Mamoru Suzuki (Osaka Univ.), Fumitaka
Makune (Tokyo U.)