2011/9/26

Cheiron School 2011

Protein Crystallography
Macromolecular crystallography (MX)SPring-8 / JASRI
Structural Biology Group

Takashi Kumasaka
http://bioxtal.spring8.or.jp
E-mail: kumasaka@spring8.or.jp

1

Contents• Introduction• Data collection method
Experimental error, Detector• Synchrotron beamline
Microbeam beamline, Beamline automation,
Remote data collection,…

• Radiation damage
Mechanism, Cryo technique, …

• Anomalous data collection
Optimum wavelength, Estimate signal, …
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Introduction

3

Structural study of Ribosome
Produced
proteinRibosome …
plays a factory to produce protein from genetic information
Central Dogma
DNA -> RNA -> Protein
A major target of antibiotic drug

Ribosome

RNA (gene)To reveal function from structure
It is considered that ribosome is difficult to be crystallized
because of its huge size. Prof. Yonath started its trial from 1980’s
when the other prize winners (Profs Steitz and Ramakrishnan) did
not undertake it.
> Structures determined at 2000.
Used synchrotron
ESRF ID2, ID14-2, -4, ID29
APS 19-ID
CHESS F1
DESY BW6 & BW7B
Prof. Ada Yonath
PF BL6A… and many others
2009 Nobel Prize

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Protein structure

Building block: 20 amino acids

5

6

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Application to drug
discovery

Marked results

EnzymeReaction selectivity < Key and keyhole

Relenza (Influenza)
Only works a key (chemicals) can entry
into the keyhole (enzyme)

Drug Design: Chemicals recognize and bind to protein
structure to regulate protein function.
•National interests and drug discovery.
•Keen competition in drug development.

Indinavir (AIDS)•Importance structural analyses of drug-target proteins

7

Crystal structure and synchrotron
Number of determined structures

Asian contribution
1000
PF (1993~)

900

SPring-8 (1999~)

800

Asia

700
600
500
400
300
200
100
2007

2006

2005

2004

2003

2002

2001

2000

1999

1998

1997

1996

1995

0

Nowadays, most structures are determined using SOR.
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Determination of important and complex
structures
Supramolecular complex

kDa (Mw)

3G SOR came into this field from 2000,
and accelerates large molecule
analysis.

Biologically important proteins
including membrane proteins:
Calcium pump, Rhodopsin,
Bacterial flagella, Drug effilux protein
9and so forth.

Crystal structure and synchrotron
Number of determined structures

Nowadays, most structures are determined using SOR.
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Determined Structure / yr

Microbeam

Ribosome

Automated Software

Hours

6500

2000
0.001mm30.0001mm3
150
2010

2000

minutes

1990

HIV protease

Membrane protein

1980

1970

1st SOR use

Enzyme structure

1st structure

1958
1960

Sample size

0.01mm3

CCD detector

Days ~

3G SOR

Anomalous phasing

0.1mm3

Imaging plate detector

Week ~

2G SOR

Cryo technique

2D detector

Measurement time

History of development in MX

11

Advances in Protein Crystallography by Synchrotron Radiation
Steps in crystallographic analysis
Samplepreparation

Before SOR

After SOR

3G SOR

Small amount of samples for
10 mg~

0.1 mg~Small crystalline size

Crystallization

Diffraction
Measurement

0.1 mm3~

0.01 mm3~0.001 mm3~

High speed data collection
day ~ week

20 min ~5 min ~

New phasing method

Phasing
Structure
Refinementmonth ~ year

Day ~ a few months

Automated refinement by high resolution data
Month ~ year

Day ~ weeks

Synchrotron data collection> effective to not only X-ray measurement but also all other exp. steps
in scale down / time reduction / high resolution.

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Overview of data collection method
Experimental error, detector…

13

Data Collection
Real Space

The Principle of X-ray
Crystallographyf (x y z)
Atomic Scattering Factor(Real Number)
CrystalReciprocal SpaceF (h k l)
Structure Factor(Complex Number)

X-ray Diffraction
Fourier TransformF(hkl) =  fj exp[2i(hx+ky+lz)]

Computation
Atomic Coordinate

Diffraction Spot 14

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To take good quality data sets
Essentials in high quality data collection:
Incident X-ray: Intensity, Divergence, Wavelength
Detector: Detection accuracy, Speed, Image resolution
Crystal: Crystalline order, Size, Radiation resistance15

Experimental setup
Laboratory
Rotating anode(Cu, etc)

Detector

Metal foil filter(Ni etc)
SpecimeneX-ray mirror
Filament

Collimator,
Slit

SOR
e-

Synchrotron

Monochromator
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Parameters in oscillation method
Detector rotation
Cameradistance

Beamdivergence1/(Ewald sphere)

Crystallographic parameter
Lattice constant, Crystal orientation

Rotation axis

Direct-beam position
17

Signal-Noise Ratio (S/N)
Signal:Noise:

Diffraction intensity ~ Dose dependent
Radiation damage ~ Dose dependent
Scattering noise ~ Dose dependent
Detector dark noise ~ Time dependent
Detector readout noise ~ Image number dependent

Intensity

Signal

Noise
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Mosaic spread

Spot sharpness depends on crystalline order.
19

Unit cell repetition & Intensity ~ Laue function
82

62

42
22
20

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Cell repetition and Mosaic spread
I: Peak height of a spot profile
N: number of mosaic crystals
V: average size of mosaic crystals

Half size in each dimension
Type 1:
Small numberof mosaic crystals

Type 2:
Small sizeof mosaic crystals

N1 = N / (23)V1 = VN2 = NV2 = V / (23)I1 = I / (23)I2 = I / (22*3)

21

Diffraction power of crystal
Darwin’s FormulaE (h) I03e 4 P  L  A  Vx
2

F
(
h
)

V2
m2c 4

I0: Incident intensity, : Angular velocity of crystal rotation, : Wavelength,e: Charge of electron, P: Polarization factor (= (1+cos22)/2),
L: Lotentz factor (= 1/sin when spindle x-ray),
A: Absorption coefficient, Vx: Crystal volume, V: Lattice volume
In case of protein crystal…

• High solvent contents (25 ~ 75%)
• Large unit cell
> Weak diffraction power ~ Low resolution

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Crystal packing ~ molecular vibration ~resolution
Vibration in solution > Movie
Packing density VM:
VM = Vcell / Mwcell

4

Resolution (Å)

Relationship with B-factor (DWF)

y=0.048x+0.646
R=0.882
B-factor (Å2)

High density (small VM) > High Rigidity

0

10 20

30 40

50 60

70

80

Packing control by humidity control
FMS (Free Mounting System)> lower humidity around crystal> dehydration> induce phase transition(Kiefersauer et al., 2000)

Resolution (Å)(Kantardjieff & Rupp, 2003)

VM (Å3/Da)
0

1

2

3

4

5 23 6log<I>

Relative Wilson statistics and intensity

Detection
Limit

Resolution Limit

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Resolution limit

Resolution and incident intensity

Incident photon
K. Hirata (RIKEN), Personal communication25

Diffraction power reduction
by radiation damage

K. Hirata (RIKEN), Personal communication

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2D detectors for MX
CCD

CMOS
Area size(100-400mm)
Resolution(50-100m)

Silicon
Pixel

IP
◎

○

△

◎

○

ME+FOTcm sq. orderby processing tech.

ME

◎

○

◎

○

△

○

Few-200 m
Phosphor

10 - 100 m
FOT&phosphor

Few m100~200 m~200 m

50 m~

Noise

Cost

Amorphous
Selenium○

Sensitivity

Dynamic
range

DirectMulti-element

Readout
Speed

Skew

Indirect

◎

○

○

◎

△

Sub mSecContinuous
readout

Sec

Sec

Real time
Counting

Min

△～◎

△～◎

△

△～◎

◎

Phosphor &Window

PhosphorLow for high E
photon

△～○

○

△

◎

△

Relatively highreadout & dark
noise

Successful Cooling
Phosphor/FOT/Window

Higher noiseby polycrystalline

Counting(counting loss athigh dose)

Stray light of laser /Loss of fluorescence
Capture

◎

△

◎

◎

◎

○～◎

FOT

DirectDirectDirectGeometry at
readout◎~20 bit

△

○

○

◎

~12bit~16 bit~16 bit∞ (Counting)

◎

△～○

◎

？

△

◎

Versatile
Processing
technology

ComplexsystemCheap
but small

Expecting Future
development

Original tech. andmonopolistic

Simple andmatured technology

27

Imaging plate
Plastic X-ray sensitive film
Photostimulated luminescence by BaFBr:Eu2+

Exposure Position of IP

Erasing Lamp

Erasing Position of IP
Reading Position
of IP
PMTs
Double-BeamHelical-ScannerSR
3 IPs Transport(Steel-Belt)

50mW laser(Rigaku, Japan)

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CCD Detector

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CCD detector system

30

Area Detector Systems Co. http://www.adsc-xray.com

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Silicon pixel detector

31

Dectris: http://dectris.com/

Continuous rotation data collection
by CMOS Detector

Hasegawa (JASRI) &Yamamoto (RIKEN) et al.
Hamamatsu Photonics

Crystal
X-ray
Shutterless
Goniometer
Rotate with a constant speed

Read out images
with a constant frame rate

High throughput and/or Fine slice data collection
Specification

Hamamatsu C10158DKADSC Q210

Scintillator

CsI:Tl

Gd2O2S:Tb

Pixel size [mm2]50 x 5051 x 51

Detector area [mm2]118.8 x 118.8

210 x 210

Output data [bits]

14

16

Dynamic range

6,000

14,10014 msec / pixel

1.1 sec / frame

Dead time due to readout

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Synchrotron Beamline

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3: Recent advances in MX beamlines
MX Beamlines at SPring-8

 9 of 53 beamlines are dedicated to MX

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Beamlines and User
Accessibility
1. Public Beamlines (BL41XU, BL38B1; JASRI)
Academic use + Proprietary use (incl. Mail-in service)
2. Contract Beamline (BL44XU; Osaka Univ.)
Academic use
Contract Beamline (BL24XU; Hyogo Pref.)Academic use + Partially opened to proprietary use
3. RIKEN Beamlines (BL26B1&B2, BL32XU; RIKEN)
RIKEN’s academic research + Partially opened to public use (20%)
4. Pharmaceutical Industrial Beamline (BL32B2; PcProt)
Fully operated for propietary useby the members ofJapan Pharmaceutical Manufacturers Association (JPMA)
35

Synchrotron MX
Brilliant synchrotron radiation facilitates MX research
1. For cutting edge researchHigh precision data collection
for Micro-crystal & Large unit-cell samples
2. For structural genomics approach
Automated and rapid data collectionfor High throughput screening
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SPring-8 MX beamline complex
Micro Focus(BL32XU)

Large Molecular Complex(BL44XU)

・Micro Focus Optics for Micro-Crystal< 10 μm
・Sample Handling for Micro-Crystal• Parallel Beam for Large Unit Cell
> 500 ÅMicro DiffractometerP2 station
for Virus

Micro Focus Optics

Remote Access

High-speed Network

High Throughput(BL26B1/2,
BL32B2, BL38B1)

High-resolution Analysis(BL41XU)• High Precision Data collection• Sub-atomic resolution

・Stable bending
magnet beam・Beamline automation
・Mail-in Data
Collection
2010/07/09

Data Server

Changer

Visualization of H-atom・Large Data Storage
・On-line Analysis
Diamondbase– SPring-8 Joint Workshop
2nd ・Data

Substrate Complex

37

Industrial Applications of Synchrotron Radiation

Get more structures and details
Resolution More precise structure analysis
Visualization of Hydrogen

0.06nmBL41XU High‐flux Beamline

AtomicAutomation of
Structure determination

0.12nmHigh‐throughput
BL26B1/2, BL38B1

Amino-Acid

BL32XU Micro‐beam Beamline
Open the door for
Structure determinationfrom Micro-crystals

nm
Molecule

1nm

100m

50m

20m

1m

Crystal Size
38

38
2008/08/07 山本雅貴

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Development of micro-beam beamline
X-ray crystallography of proteins related to human disease and aging.
Micro-beam optimized for Micro-crystal
Standard>50m

Micro-crystal<10m

Current Limit
20~30m

50m

50m

50m

Target Crystals
・Beam Size
・Flux density

Current
30×30
109Target Beam Size
1×1
m2
10
>10 photons/sec./m2
39

Beam profile of SPring-8 BL41XU

R&D target for Micro-crystallography
Micro-crystal･ Small size crystal（<10mm）･ Weak signal（106copies）

Maximize signal-to-noise ratio

・Generate micro-beam・Optimize experimental equipments

Generate Micro-beam･ Stabilize micro-beam･ Optimize beam size

Micro-beam

Optimize experimental equipments
・Crystal handling・High-precision goniometer・Reduce background noise・High-sensitive detector
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Design concept of BL32XU

1. Brilliant source
2. Simple components
3. Focusing X-rays with large magnification factor
4. Changeable beam size at sample position

41

K. Hirata, M. Yamamoto (RIKEN) et al.

Beamline components

Hybrid in-vacuum undulator

Front end

High precision double crystal monochromator

sampleK-B focusing mirrors

Co-axial sample camera

High precision goniometer

High efficient CCD detector

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EEM-mirrors for 1 um focusing

Designed mirror surface shape

Kirkpatrick-Baez Mirror

50

Horizontal Focusing Mirror : f=0.93m
Vertical Focusing Mirror : f=1.41m

Mirror shape :Elliptical

Mirror length :400 mmEnergy range :8-20 keV

20

Mirror material :

SiO2

10

Mirror surface :Pt-coated

Glancing angle :3.5mradHeight (m)

40
30

0
0

100

200

300

400

43

Position (mm)Design of focusing optics• Virtual light source is TC-Slit (located at 36m upstream of 1st mirror)• Pt-coated elliptical mirrors with K-B (Kirkpatrick-Baez) configuration• Magnification factors: 26 in vertical, 40 in horizontal• Beam divergence at sample position < 2 mrad
• Available X-ray energy range: 8 - 20 keV, especially high-flux at 12.4 -13.8keV

ID

FE DCM30m

TC slitK-B mirror

sample

0.73m

46.6m
84.5m

Beam size @ sample

1(H) x 1(V) m2

20(H) x 19(V) m2

TC slit size

40(H) x 26(V) m2

800(H) x 500(V) m2

Photon flux@12.4 keV

6 x 1010 photons/s

2 x 1013 photons/s

Glancing angle is designed at 3.5mrad

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Achieved beam size（2009/11/27）

Intensity / arb. unit

1.0

FWHM
0. 9 m

0.8
0.6
0.4
0.2
0.0
-3

-2

-1

0

1

2

Vertical beam profile
Intensity / arrb. unit

Horizontal beam profile

3

1.0

FWHM
0.9 m

0.8
0.6
0.4
0.2
0.0
-3

-2

-1

0

1

2

3

Position / um

Position / um

Focused photon flux : 6.2x1010 photons/sec
The smallest & highest flux density in the world
45

Micro-crystal diffractometerHe cryo-cooler

X-ray CCD detector

Air-bearing goniometer
Coaxial microscope

He Chamber
Sample changer robot
SPACE

Granite table
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Air-bearing goniometer•High-precision spindle axis with air-bearing unit
•Hi-speed rotation useful for fast centering, inverse beam geometry etc.

Eccentricity of goniometer

Laser displacement
meter

0.5
0.0

SOC(um-0.5-1.0~0.1 m/100°-1.5

Spherical steelball

Eccentricity < 0.7m/360°

-2.0
-2.5
0

50

100

150

rotation(d
eg.)
200
250

300

350

47

400

（KOHZU PRECISION Co., LTD.）

Tentative diffractometer setting

Focusing mirror -> Ion chamber -> Shutter -> Co-axial sample camera ->Collimator -> Back light -> Beam stopper
48

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The first crystal onto the 1m beam

20 m

Lysozyme
49

The first diffraction image (09/12/04)
CrystalBeam property
Exposure time
Resolution limit

Lysozyme 5um crystal
1mm square, 2.6 x 1010 photons/sec.
1 sec.
2.0 Å

Larger beam divergence did not badly affect diffraction profiles
50

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Data collection limit by crystal size
Acta Cryst. (2008), D64, 158-166◆

Protein crystals□

Small molecule crystals

Formula of diffraction powerS  ( F000 / Vcell ) 2  3  Vcryst
We collect a 2 Å resolution datafrom 2 um lysozyme crystal.

Protein crystal @ BL32XU

51

A recent result of a structure
determination
2.6Å C2221,
1 degree x 90 images,
from a single crystal(0.015 x 0.012 x 0.012 mm).
1μm beam, 1 sec exposures
Completeness=96.5%(93.9%, 2.69-2.6A)
Apr. 2010
Drs. Sengoku and Bessho(RIKEN)
Extra electron densityof a drug candidate.
52

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BL Automation

Samples & data management
D-Cha (Database for Crystallography with Home-lab. Arrangement)
Crystal information database, Schedule editor, Image browser, etc. on the Internet

53

SPACE

SPring-8 Precise Automatic
Cryo-sample Exchanger
Sample container

Sample Pin

52 pins/puck(polyoxymethylene)

0.2mm

Opposite threads
10mm

25mm
Size: 75x75x50 mm^3

SPACE

Goniometer

SampleArm

LN2 Auto-supply Chamber
Ueno, Kanda, Ida, Kumasaka, Yamamoto (RIKEN) et al.

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New attachmentfor Hampton/SPINE pinswith Unipuck and other flat type cassettes

Unipuck tray

Hampton pin(Murakami, Ueno, Yamamoto et al.,Patent #2009-115652)

SPINE pin55

from Robo-Sync web

Remote AccessConvenient use without visitation
Automation of beamline control

Mail-in data collection
 Send crystal to SPring-8
 Edit measurement condition with
D-Cha
 Collect data with a help of BL
operator

Beamline database D-Cha
Sharing information between users’ lab &SPring-8• Crystal related information• Measurement condition & schedule• Recorded images

Users’ Laboratory
LAN
Beamline control software BSS

Internet

 Integrated control of beamline optics and diffractometer.
 Automatic set-up of measurement condition.
 Multiple measurements proceed in the scheduled order.

Automation of sample mounting
Gonio

D-Cha

Web interface of D-Cha

BL control PC

SPring-8

CCD

SPACE

Sample changer SPACE Mount specimens on goniometer instead of users.
 Combination of BSS & SPACE enables automatic
data collection with exchanging crystals.

Measurement schedule

Diffraction image

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Remote access at SPring-8 MX beamlines
Basic concept
Make use of sample changer SPACE, integrated beamline control
software BSS and beamline database D-Cha
to make all operations required in MX data collection from remote
env.
Characteristic features of remote access at SPring-8• Spec fulfilling the strict radiation safety regulation in Japan− Not use the NX client for secure access to BL inside gateway.
− Remote client & local server architecture with the original
protocol.
− Authentication gateway and operation restricts units are installed
between the client-server to ensure safety of remote access.
• New original protocol developed as a SPring-8 standard
For the future use other than MX BL…
Remote access composed of three session (network connection)
1. Streaming session
2. Device control session <<<3. Result view session
57
Hasegawa, Ueno, Furukawa et al. (2010)

Streaming session
F.W.
Streaming of sample

Video image

ffserverStreaming server

F.W.

Streaming session
ffmpegEncode microscope images

Delivery of video streaming used for sample alignment
Client GUI
softwareUsers lab.

The Internet

SPring-8 OA LAN

SPring-8 Beamline
Access from the out side is restricted58

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Device control session
Control command is transferred through
F.W.
F.W.
the authentication gate way and operation restrict unit
ffserverStreaming session

Command filteringffmpegControl commands are transferred
only to the assigned BL
only during the assigned BT

Control
command
Client GUI
softwareDevice control session
Authentication
GW
Proposal
DB

Users lab.

Authentication using SSL
Correct combination of• client certificate (sent by e-mail)• pass phrase (sent by physical mail)is needed to establish connection

Operation
restrict unit

Control
commandDevice server
(BSS)
BL-WS

Monitoring hutch status
Cannot control devices unless thehutch is normally closed.

Robot server
DetectorserverSPring-8 standard protocol
BL interlock

59

Diffractometer &sample changer

Result view session
Session to check and download recorded data
F.W.

F.W.

ffserver

Client GUI
softwareffmpegControl
commandAuthentication
GW•Diffraction images•XAFS spectrum

Streaming session

Device control session
Operation
restrict unit

Device server
(BSS)
BL-WS

ProposalDB

Measurement
Condition

Robot server

Users lab.

Web Server
D-Cha
Result view session• User info.

Make use of D‐Cha • Sample info.
developed for mail‐inDB•Diffraction images•XAFS spectrum

DetectorserverDatastorage60

Diffractometer &sample changer

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Client GUI
software

A Windows application coded by
Python2.6.
Distributed as a binary executable
61

Radiation damage

62

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Radiation damage
Bacterial flagelin F41 Crystal@ SPring-8 BL41XU

1st frame

15min.

Very Thin Crystal(≒10m)25min.

Total Flux at Sample ≒ 1013 photons/sec/mm2
F.A. Samatey, K.Imada, S.Nagashima, K.Namba (ERATO, Osaka Univ.)

63

Interaction between photon and protein
Primary Effect・Absorbed photon energy > Temperature increment
・Photoelectron formation> Chemical reduction / Reactive radical formation
X-ray dose
dependent
Temperature / Time independent

Secondary Effect・Chemical reaction by free radicals
X-ray dose, temperature / time dependent
64

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Photon-electron interaction

PE: Photoelectric absorption, R: Thomson (Reyleigh) scattering,
C: Compton scattering
65

Cryocrystallographic technique
Suppress thermal degradation of sample
diffusion and reaction of free radicals
at cryogenic temperature (30 – 100 K)
using cold N2/He gas stream

Cryostat

Sample Mount Pin & Cryoloop

66

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Radiation induced temperature increment
under cryogenic condition
I0 = 1×1014 photon/sec/mm2
170h=100W/m2/K

160

Adiabatic

150

100 m thick layer

T(K)40K/Sec
140

100×100×100 m3 Cubic130T = +20 K

120

100×100×10 m3 Plate110

T = +6.5 K

100
0

1

2

3

4

t (sec.)

5

6

7

8

Heat transfer speed depends on crystalline size and shape.
Kuzay et al. Acta Cryst. (2001). D57, 69-81

67

SPring-8 BL41XU

h=100W/m2/K
I0 = 1×1014 photon/sec/mm2

Adiabatic

Adiabatic

Glass transition temp.
I0 = 5×1013 photon/sec/mm2
SPring-8 BM BLs

Adiabatic

I0 = 1×1013 photon/sec/mm2

Kuzay et al. Acta Cryst. (2001). D57, 69-81

68

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Phase transition of water

Water structure and science by Martin Chaplin 69
http://www.btinternet.com/~martin.chaplin/

Reactive radical formation by photoelectron production
Water
h
+
H2O
H2O + + H2O
e+ H2OH2O - + H2O

H2O + eH+
+ OH・
H2O H・+ OH-

H2O
Disulfide bridge(p)-CH2SSCH2-(p)(p)-CH2SSCH2-(p) + OH・(p)-CH2SS・+CH2-(p) + OH(p)-CH2SSCH2-(p) + OH・
Cysteine(p)-CH2SH + e(p)-CH2SH + H
Aspartate & Glutamate(p)-CH2CH2COO(p)-CH2C・+ H2COOTyrosine(p)-CH2C6H4OH(p)-CH2C6H4OH・+
Methionine(p)-CH2CH2SCH3 + 2H・

H・+ OH・ Hydroxyl radical
Half-life ~10-9 s(p)-CH2SS・+CH2-(p) + e(p)-CH2SS・+CH2-(p) + OH(p)-CH2SOH + (p)-CH2S・(p)-CH2SOH + (p)-CH2S・(p)-CH2 + SH(p)-CH2 + H2S(p)-CH2C・+H2COO- + e(p)-CH2CH2・ + CO2(p)-CH2C6H4OH・+ + e(p)-CH2C6H4O・ + H+(p)-CH2CH3 + CH3SH

70

Burmeister, Acta Cryst. (2000). D56, 328-341

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2011/9/26

Estimation of radiation damage
SampleHen egg white lysozyme

Space group

P4３212

Latticea = 78.54 , c = 37.77 ÅBL

SR (SPring-8 BL45XU)

Data set11(1 ~ 11)

1 (12)

image/set

95

245

Wavelength

1.02Å

1.02Å

Oscillation angle 1.0°

1.0°

Camera distance

150mm

220mm

Exposure

5 sec

60 secDetector

Jupiter210(CCD)RAXIS-V (IP)N. Shimizu (JASRI) et al.

71

Variation of I/σI and Rmerge
0.060

40

I/σI
0.050
30
I/σI

0.040

R merge20
0.030

Rmerge0.020

101Exposure

2

3

4

5

6

7

8

9

10 11

(# data set)72

36

2011/9/26

Variation of lattice constants
(Å)

(Å)
36.96

78.80

a,b axes

78.78

36.94
36.92

78.76

a,b

c
36.90

78.74

36.88

78.72

c axis

36.86

78.70

1

2

3

4

5

6

7

8

9

10 11 (#data set)

Exposure
73

Radiation damage in real space
Data set

electron density at 1.6 Å resolution

１

Gln121

R-factor 18.51 ％R free20.64 ％

fo-fc

2.5σ

Cys94

fo-fc －2.5σ
2fo-fc1.0σ

Gln121

12
R-factor 18.50 ％R free20.68 ％

Cys94

74

37

2011/9/26

Dose limit
Estimated dose limit for ionizing radiation

3 x 1017
1 x 1016

keV/mm3 (~ 5 x 107 Gy)
photon/mm2 @ 12.4keV（Henderson, R. (1990). Proc. R. Soc. London Ser. B, 241, 6-8.）

4 x 1017

keV/mm3（Gonzalez, A., Nave, C. (1994). Acta Cryst. D 50, 874-877.）

5 x 1016

photon/mm2 @ 12.4keV（Sliz, P.,Harrison,S.C.Rosenbaum,G., (2002). Structure, 11, 13-19.）

75• Use of a helium gas cryostream lowers the sample
temperature to around 35 K.
He-cryo• Many works reported the effect of the lowtemperature below 100K.

N2-cryo• The behavior of the generated hydrogen gaschanges depending on the temperature?

BL41XU at SPring-8(Alke Meants, RD5 (2008), PSI)

• Use of as high X-ray energy as possible.

The damage is generally thought to decrease at higher X-ray
energy as the absorption is lower.
• Dose control

38

2011/9/26

Raddose* GUI for SPring-8 BL(Kawamoto (JASRI) et al.)Beam size and thickness of the attenuator arechosen from a pull down menu.
Photon flux (PHOSEC) is automaticallycalculated by parameter tables of the flux, thebeam size and the attenuator against
wavelength.
Although the number of monomers in theasymmetric unit is inputted, it is changed into thenumber of monomers in the unit cell (NMON) by
using CCP4 libraries.

As for the percentage for dose limit, the value pera irradiation point is displayed.
The original log file of RADDOSE is displayedwhen “View full log” is checked.
*) Murray, J. W. et al. (2004). J. Appl. Cryst., 37, 513-522.

In case of a membrane protein:Ca2+-ATPase in various states
E1·2Ca2+

C2
1.95 Å

E1·ATP(AMPPCP)

C2
2.5Å

P21
2.9Å

E1P·ADP(AlF4-·ADP)

C2
2.4Å

E2·Pi
(MgF42-)E2(TG,BHQ,CPA
)

P21
2.3Å

P41212
2.1Å

C2
2.9Å

P21
3.3Å

Courtesy of Prof. C. Toyoshima (Univ. Tokyo)

78

39

2011/9/26

cross-sections of an
E1·2Ca2+ crystal

79
Courtesy of Prof. C. Toyoshima (Univ. Tokyo)

Data acquisition tools
Multiple X-ray exposures on one/several crystals
to control serious radiation damage during data collection
to find better diffracting crystals or crystal segments
・Vector Centering：・Multiple Centering：

Changing exposure position linearly in every
constant frames on One crystal

Multiple exposure positions using several
crystals in One cryo-loop

25m

①
②

③
⑥
④
⑤

25m

80

40

2011/9/26

Anomalous data collection

81

Harker Diagram ~ Structure factor and phase

F (h) | F(h) | exp i

Im
i|F(hkl)|  f j exp 2ih r j

F(hkl)

Complex spaceO-|F(hkl)||F(hkl)|Re-i|F(hkl)|
82
exp i = cos  + i sin412011/9/26

Isomorphous replacement
Im| FP |: Native| FPH1 | : Derivative 1
FH1


O

| FPH2 | : Derivative 2
Re FP = | FP | exp i

FH2
H1
H2

FPH1 = FP + FH1

83

Anomalous Phasing
Anomalous EffectAnomalous term of zinc (Ze = 32)
6

f”

f”

4
2

f-

0

Electrons

f+

-2

f0
f’

f± = f0 + f’ ±if”

-4
-6

f’

-8

Smaller than usual
heavy atom effects↓
Need high quality data

-10
-12
-14
0

0.5

1

1.5

2

Wavelength

2.5

3

84

42

2011/9/26

2 Wavelength MAD| F |: Native| F (h+) | : Anomalous| F (h-) | : Anomalous| F (h+) | : Anomalous| F (h-) | : Anomalous
FP=| FP |exp i

Single phase solution
can be determined.
85

Incident wavelength and anomalous signal
Oxidation state and XANES spectra
for SeHolden & Nadeau, 2008

Anisotropic anomalous scatteringfactors f′ and f′′ for Se in SeMet

Schiltz & Bricogne, Acta Cryst D64, 711 (2008)
86

43

2011/9/26

Recent trend of isomorphous phasing

87

SAD| F |: Native| F (h+) | : Anomalous| F (h-) | : AnomalousPhase probability function shows
bimodal.
>> Phase improvement by density
modification>> High precision data collection
88

44

2011/9/26

Estimation of anomalous signal
0

Dauter, Acta Cryst. D62, 867 (2006).

89

Phasing power ~ NCS / %Solv

#NCS

%Solv
10

20

30

40

50

60

70

80

90

1

1.11

1.25

1.43

1.67

2.00

2.50

3.33

5.00

10.00

2

1.57

1.77

2.02

2.36

2.83

3.54

4.71

7.07

14.14

3

1.92

2.17

2.47

2.89

3.46

4.33

5.77

8.66

17.32

4

2.22

2.50

2.86

3.33

4.00

5.00

6.67

10.00

20.00

5

2.48

2.80

3.19

3.73

4.47

5.59

7.45

11.18

22.36

6

2.72

3.06

3.50

4.08

4.90

6.12

8.16

12.25

24.49

7

2.94

3.31

3.78

4.41

5.29

6.61

8.82

13.23

26.46

8

3.14

3.54

4.04

4.71

5.66

7.07

9.43

14.14

28.28

9

3.33

3.75

4.29

5.00

6.00

7.50

10.00

15.00

30.00

10

3.51

3.95

4.52

5.27

6.32

7.91

10.54

15.81

31.62

15

4.30

4.84

5.53

6.45

7.75

9.68

12.91

19.36

38.73

20

4.97

5.59

6.39

7.45

8.94

11.18

14.91

22.36

44.72

25

5.56

6.25

7.14

8.33

10.00

12.50

16.67

25.00

50.00

Arnold & Rossmann, PNAS 83, 5489-5493 (1986)

90

45

2011/9/26

S-SAD Phasing of Thaumatin
Thaumatin (miracle fruit)
Mol. weight

22.132 Da

Amino Acids

206 residues

Sulfurs

Met : 1
Cys : 16 ( S-S : 8 )12.1 res. / 1 Sulfur

Space group

P41212

Solv. Content

0.54

Experimental conditions
Crystal size

0.20 mm x 0.10 mm x 0.05 mm

Beam size

0.05 mm x 0.05 mm

Exposure

1 sec. exposure, 1deg. oscillation, 360 frames

Software
HKL2000(+SCALA )+ SHELX C/D + SOLVE/RESOLVE + ARP/wARP

M. Kawamoto (JASRI, SagaLS) et al.

S-SAD Phasing of Thaumatin

1.10 Å

1.30 Å

1.50 Å

1.70 Å

1.90 Å

Distancce

150 mm

150 mm

110 mm

110 mm

110 mmCell

a = 57.873,
c = 150.186 Å

a = 57.821,
c = 150.142 Å

a = 57.985,
c = 150.393 Å

a = 57.895,
c = 150.247 Å

a = 57.845,
c = 150.137 Å

Resolution

50 – 2.1 Å
0.032 / 0.046(0.051)0-0 – 45º(multi.≒1.9)
0 – 60º(multi.≒2.5)

Ranom / Rpim (Rmerge)# found sulfurs
Seq. Cov. (Rwork/Rfree)

0.026 / 0.038(0.049)13
4 % (0.303 / 0.624)

0 – 90º(multi.≒3.8)
0 – 120º (multi.≒5)0 – 180º(multi.≒7.5)

・Model constructed
・Model constructed in part・Substructure determined
・No solution

0.012 / 0.019(0.059)0-0 – 240º(multi.≒10)
0 – 360º(multi.≒15)

0.009 / 0.013(0.047)0-0.011 / 0.017(0.060)11
41 % (0.305 / 0.562)

0.014 / 0.020(0.040)00.013 / 0.017(0.043)13
27 % (0.305 / 0.575)
0.011 / 0.015(0.044)14
99 % (0.222 / 0.280)
0.010 / 0.014(0.051)
15
99 % (0.227 / 0.299)

0.018 / 0.023(0.037)00.016 / 0.022(0.043)
1140 % (0.303 / 0.612)
0.015 / 0.020(0.051)
10
94 %(0.280 / 0.365)

0.020 / 0.028(0.055)12
4 % (0.299 / 0.588)
0.019 / 0.025(0.063)
12
99 % (0.208 / 0.257)

0.013 / 0.015(0.057)12
99 % (0.206 / 0.222)

0.016 / 0.019(0.071)
14
99 % (0.192 / 0.243)

M. Kawamoto (JASRI, SagaLS) et al.

46

2011/9/26

S-SAD phasing of Lysozyme@ SPring-8 BM BL
14,307 Da
129 residues
・Sulfurs Met: 2
Cys: 8 (S-S: 4)
12.9 res./1 sulfer
・%Solv 0.37
・Mw

・Resolution (Å)
50.00-1.08 (1.12-1.08)
・Space group
P43212・Unit cell (Å)
a = 78.88, c =36.93
・Redundancy
26.4(11.9)
・Observations
2432608
・Unique reflections 46104
・Completeness (%) 96.4 (79.2)
・Rsym (%)
2.4 (36.2)
・I/σ(I)
95.56 (8.88)

・Crystal size (um)・Wavelength (A)・Oscillation range
・Oscillation angle
・Exposure time
・Camera distance

200 x 180x 100 30.85Å
720 deg.
0.5 deg.
1.5 sec
120 mm・Data processing
・Phasing

HKL2000
SHELXC/D/E

・Model Building

ARP/wARP

Anomalous scatteringfactor of sulfur(Å) f’f’’
5.01 -7.4614 4.1039
2.50 0.3564 1.3314
1.50 0.3115 0.5298
1.30 0.2689 0.4047
1.00 0.2206 0.2937
0.85 0.1509 0.1771
93
S. Baba (JASRI) et al. @ BL38B1

Fall-off factor

Damage control in MAD data collection

t
0,1,2
……
……
 = 00,1,2 1…… 2 ……3,4,5

0,1,2 ……

No Wedge
Wedge
=1
3

Almost perfect isomorphism
Non-isomorphism
by crystal degradation
94

47

2011/9/26

Overview of Beamline BL45XU

Trichromatic concept for optimizing MAD-experiment• Tandem vertical undulator (for High Quality Beam)• Trichromator (for Rapid Data Collection)
Yamamoto, Kumasaka, Fujisawa, Ueki (RIKEN) (1996)

95

Trichromatic Concept for MAD-experimentThree-wavelengths data
will be taken simultaneously for

Trichromator

1st Pair (λ1)
RemoteBeam ChopperProtein Crystal

the identical protein
crystal without changing thesetting by “Trichromator”.

Change Beam Chopper

2nd Pair (λ2)
PeakChange Beam Chopper

3rd Pair (λ3)
Edge

To improve MAD data,
inverse beam is also applicable.

Change Beam Chopper & Next Oscillation Range(3rd Pair to 1st Pair)

96

48

2011/9/26

Bacterial Chitosanase (Mw 34k, 7 SeMet)
Source: Gram-Negative Bacterium (Matsuebacter chitosanotabidus 3001)
Function: Hydrolysis of glycosidic bonds of chitosan(○GlcN-GlcN, GlcN-GlcNAc, GlcNAc-GlcN, ×GlcNAc-GlcNAc)

P212121a = 51.5, b = 56.2, c = 206.8 Å
1.7 Å Resolution
Two monomers / Asym. unit

97
Kumasaka (RIKEN) et al., 2002

Effect of Trichromatic Data Collection
Data Collection Statistics
Data
Cho1: Remote

Observations

Individuals

I/

RmergeRiso

B

260,40265,579

18.5

0.049

－

－

Peak

269,82166,482

17.4

0.053

0.057

0.08

Edge

269,362

66,428

17.5

0.084

0.048

0.11

261,577

65,695

19.8

0.045

－

－

Peak

263,567

66,480

18.7

0.048

0.064

0.09

Edge

263,387

66,449

17.1

0.076

0.078

-0.39

Cho2: Remote

Cho1 (Trichromatic)

Chitosanase Crystal(Length=0.4 m)

Cho2 (Conventional)
98

49

2011/9/26

Effect of Trichromatic Data Collection
Variation of Fall-off factor
Cho2(Conventional)

Cho1(Trichromatic)99

Effect of Trichromatic Data Collection
Comparison with dispersive Patterson maps
Cho1 (Trichromatic)

Cho2 (Conventional)

100

Harker section (w = ½)50

2011/9/26

Effect of Trichromatic Data Collection
Phasing Statistics (20 – 1.7 Å)
Data

Cho1 (Trichromatic)
RemoteRCullis (iso)#RCullis (ano)Edge0.82 /
0.84

0.83 /
0.88

0.91

0.99

0.94

Lack of closure(iso)#
Lack of closure
(ano)

8.98

Figure of merit0.6057

Remote0.94

8.9 / 14.0

8.1 / 12.5

16.56

7.32

1.22 /
0.81

1.19 /
0.82

8.11

Peak

Edge

0.78 /
0.83

0.76 /
0.86

0.91

0.99

11.4 /
14.7

10.3 /
16.8

15.91

6.37

1.40 /
0.90

1.38 /
0.89

0.6167

Phasing power#<||>*

Cho2 (Conventional)

Peak

44.2

(33.9)

47.8(39.4)#: Acentric and centric values before and after slash.
*: Phase difference against phases calculated from refined model
Parenthesis show the values within the range of 10-2.5 Å. 101

Effect of Trichromatic Data Collection
Phase difference  (degree)

Phase difference against true phase

Cho1

65
Cho1(Trichromatic)60

Cho2(Conventinal)

55
50

Cho2

45
40
35
30
0.03

0.1

0.17

d-2 (Å -2)

0.24

0.31
Tyr 165 (Chitosanase A-chain)
1.7 Å MAD phase 102(without any phase modification)51

2011/9/26

Other phasing method:
Molecular replacement?
Known determined
structure

Unknown
but probably similar
structure

To solve unknown structure,a known structure is used
as a approximation.
The known structure will beselected by sequence similarity.
Highest sequence similarity
might gives highest structuralsimilarity.

Rotation search?

Translation
searchHow to pack the molecules
into the cell ?
> 6-D search
103

Patterson function
Intramolecular・Intermolecular vectors
Real space(r)= S F(S) exp[-2i S・r] dS
P(u)= S |F(S)|2 exp[-2i S・u] dS

Patterson space

Integration radius

104

52

2011/9/26

Euler angles, 

z’’
x’
z
Rotation axis:
cos sin 01 0
0 cos sin 0




sin cos 00 cos sinsin cos 0




 0
0 10 sin cos  0
0 1
Order
in 3 step rotation: 3
2
1

105

Polar angles, 

z’’’’

y’’’

z’’
-y’
-z
cos sin 0cos 0 sincos sin 0cos 0 sincos sin 0






sin cos 0 0 1 0 sin cos 0 0 1 0 sin cos 0






 0
0 1sin 0 cos  0
0 1sin 0 cos 0
0 1
5

4

3
Real rotation ~ 

2

1

106

53

2011/9/26

An example of rotation function
α

β

γ

ｘ

ｙ

ｚ

Correlation
Coefficient

R-factor

30.37

54.61

351.97

0.000

0.000

0.000

16.0

48.9

59.63

125.39171.97

0.000

0.000

0.000

16.0

48.9

27.57

41.41

20.51

0.000

0.000

0.000

9.2

51.1

62.43

138.59200.51

0.000

0.000

0.000

9.2

51.1

17.43

98.67

334.32

0.000

0.000

0.000

7.2

51.7

72.57

81.33

154.32

0.000

0.000

0.000

7.2

51.7

41.73

139.11

197.95

0.000

0.000

0.000

7.7

52.1

48.27

40.89

17.95

0.000

0.000

0.000

7.7

52.1

81.84

98.18

226.67

0.000

0.000

0.000

8.2

51.6

8.16

81.82

46.67

0.000

0.000

0.000

8.2

51.6

107

Modeling & refinement of structure
Modeling: Construct molecular model to fit obtained electron
density using interactive molecular graphics software
or automated modeling software.
Refinement: Optimization of observed and calculated F databy shifting atomic coordinates.
R-factor: Crystallographic Reliability-factor

R1= ||Fo|-|Fc(r)||/ |Fo|wR2=( w(|Fo|2-|Fc(r)|2)2/ w(|Fo|2)2)1/2
Cross validation of R-factor (Rfree)108

54

2011/9/26

Refinement of structural model
1) Unrestraint refinement
Only using R-factor refinementin case of ultra-high resolutions (0.8 A or higher)

2) Restraint refinement
Coupled with molecular mechanics
Model validity is also guaranteed by low energy~ structural stability

E  Echem  wxray E xray

Target function

E xray  h FO h  kFC h 

2

109

Basics of molecular mechanics (MM)
Energy calculation of atomic bonds and interactions
by classical mechanics.
bond length
bond angle
dihedral/torsion
improper dihedral
van der Waals
Echem = Ebond + Eangle + Edihe + Eimpr + EvdW + Eelec
electrostatic
e.x. Water

Ebond = kbond (r - ride)2
Eangle = kangle ( - ide)2

1.4 A
O

104.5 deg
H

0.958 AH

1.2 A

Force Field

TIPS3P Parameter
Bond O-H
450.0
Bond H-H
0.0
Angle H-O-H 55.0

0.9572
1.5139110104.5255

2011/9/26

Summary• Signal enhancement / Noise reduction– Diffraction signal:
Crystal size, Beam flux/flux density, …
– Scattering noise
Beam size / profile, Cryosolvent, Wavelength,
Oscillation angle, Beam stop / Helium path, …
– Radiation damage:Helium cryo, Exposure time, Shift beam position,
Radical scavenger, …
– Anomalous signal
Anomalous scatterer, Wavelength, …
111

56