Cheiron School 2010

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

Takashi Kumasaka

Contents• 1: Introduction to MX• 2: Methodology of MX– 2.1: Crystallization– 2.2: Data collection– 2.3: Phasing• 3: Recent advances in MX methods– 3.1: Microbeam & Radiation damage

1: Introduction to MX
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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.

Determination of important andcomplex structures
Supramolecular complex

kDa (Mw)

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

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

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.
Indinavir (AIDS)•Keen competition in drug development.
•Importance structural analyses of drug-target proteinsminutes

2010

Hours

Anomalous phasing

Microbeam

CCD detector

Automated Software

Determined Structure / yr

Ribosome

3G SOR

Imaging plate detector

0.01mm3

2000

1990

HIV proteaseDays ~
2G SOR

Cryo technique

2D detector

0.1mm3

Membrane protein

1st SOR use

Enzyme structure

1st structure

Measurement time

Week ~

1980

1970

1958
1960

Sample size

History of development in MX

6500

2000

0.001mm30.0001mm3
150

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.

2: Methodology of MX
2.1: Protein crystallization

http://www.cir.tohoku.ac.jp/sazaki-p/%20Web_pages/Protein_crystallization.html

Hydration and packing
Dissolution:
Condensation:
Protein-water interaction
Protein-protein interaction
Water-water interaction Dehydration

Crystal packing
Nakasako, M. Philos. Trans. R. Soc. Lond. B 359, 1191-204 (2004)

Factors determining protein solubility
Protein aggregation
Electrostatic interaction ~ oriented/ordered interaction
Hydrophobic interaction ~ random aggregation
・Kosmotropic ions: multivalent anion etc.
Hofmeister series: Salting-out effectStabilizing and enhancing water-water network> Aggregate hydrophobic patches in proteins
・Organic solvent / uncharged polymer: acetone, DMSO, PEG etc.
Lower permittivity > Coulomb repulsion > aggregation
excluded volume effect in polymer > aggregation by osmotic pressure
・DetergentsHydrophilization of protein hydrophobic regions > solubility ↑・Chaotropic ions: larger ionic radii monovalent anion etc.
Guanidium ion, Urea, Iodine etc
Destroy water cluster > enhance solvent entropy
・Amino acids / polyamine
Direct interaction with protein > Reduce protein-protein interaction
・pH
Shift the surface charge in protein > modulate electrostatic interaction

Phase Diagram

http://www.cir.tohoku.ac.jp/sazaki-p/%20Web_pages/Protein_crystallization.html

Seeding technique
Optimizing crystal seed formation
Clusters

Small crystals

Single crystals

Size improvement

Macro Seeding / Micro Seeding / Streak Seeding / Heteroseeding

Type of crystal growthHigh

Driving force of crystallization

Low

http://www.cir.tohoku.ac.jp/sazaki-p/%20Web_pages/Protein_crystallization.html

Crystallization method
Use the equilibrium against concentration gradient.
•Hanging drop vapor diffusion

Vapor diffusion

Protein Solution(5~50 mg/ml,
1~10 µl)
Hanging drop
vapor diffusion

Reservoir Solution(50~1000 µl)•••

Use the equilibrium
against concentration gradientin a sealed container.

Sitting drop vapor diffusion
Dialysis method: liquid-liquid contact via semipermeable membranes.
Batch method:Simply mixing protein solution and precipitant.

Screening of crystallization condition
1. Initial Screening
Sparse Matrix Method
Screening kits available commerciallyincl. 48~50 conditions
2. Optimization
Fine tuning solution parameters: Conc., pH, additives…
1

2

3

4

5

6

A

A.S. 1.0M

A.S. 1.1M

A.S. 1.2M

A.S. 1.3M

A.S. 1.4M

A.S. 1.5M

pH 7.0B

A.S. 1.0M

A.S. 1.1M

A.S. 1.2M

A.S. 1.3M

A.S. 1.4M

A.S. 1.5M

pH 8.0C

PEG 10 %PEG 11 %PEG 12 %PEG 13 %PEG 14 %PEG 15 %pH 7.0D

PEG 10 %PEG 11 %PEG 12 %PEG 13 %PEG 14 %PEG 15 %pH 8.03. Heterogeneity check of sample solution
Impurity:
Electrophoresis, Analytical HPLC
Instability:
Enzymatic assay, Mass spectrometry
Oligomeric assembly: DLS (dynamic light scattering)From precipitants to crystals
0

7

4
Clear drop
Non-crystalline particles

Clusters

15

8

Brown aggregates
Microcrystals

2

Small crystals (0.05-0.2mm)
White aggregates

3
Transparent aggregates

96
Needles

Plates

Large crystals (0.2mm ~)Crystallization robot
RIKEN SPring-8

Dispenser robot

Microscopic image

Automated monitoring system

Tabletop dispenser robot(Thermo Scientific Hydra)

2.2: 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) = Σ fj exp[2πi(hx+ky+lz)]

Computation
Atomic Coordinate

Diffraction Spot

Crystal systems and space groups
7 systems & 230 space group

Triclinic: P1. a≠b≠c, α≠β≠γ ≠ 90°
Monoclinic: P2, P21, C2. a≠b≠c, α=β=90, γ ≠ 90°
Orthorhombic: P222, P212121,F222. a≠b≠c, α=β= γ= 90°
Trigonal: P3, P31, P3121. a=b≠c, α=β = 90°, γ= 120°
Hexagonal: P6, P61, P6122. a=b≠c, α=β = 90°, γ= 120°
Tetragonal: P4, P41, P41212, I4122. a=b≠c, α=β=γ= 90°
Cubic: P43, F432, P41312, I432. a=b=c, α=β=γ= 90°
65 Enantiomorphic space groups (which do not have a center of symmetry
due to the absence of inversions, mirrors, and glide plane elements)
out of 230 all space groups, because protein molecules do not have such
symmetry.

Bravais lattice and Laue group
Cubic (P, F, I) m3, m3m
Tetragonal (P, I)
4/m, 4/mmm

Monoclinic(P, C) 2/m

Orthorhombic(C, F, P, I) mmm
Trigonal (P) 3, 3m1, 31m
Triclinic (P) 1

Hexagonal (P) 6/m, 6/mmm

Space group
P212121

1: x, y, z, 2: ½-x, y, ½+z,
3: ½+x, ½-y, z, 4: x, ½+y, ½-z

y

x

1
2

-

1
4

1
4
1
2

+

+

+

1
2

1
4

1
2

1
4

+

+
1
4

-

1
4

+

Relationshipbetween real and reciprocal spaces
P2 (2-fold axis along y-axis through origin) > ρ(x,y,z)=ρ(-x,y,-z)F(hkl ) = V ∫Cellρ ( xyz ) exp[2πi (hx + ky + lz )]=V ∫Half − Cell

ρ ( xyz ){exp[2πi (hx + ky + lz )] + exp[2πi (−hx + ky − lz )]}= F(h kl )Other rules:
1) Screw axis shows same symmetry of its rotation
with systematic absencesex) P21, F(hkl) = F(-h,k,-l), F(0,k,0) = 0 (k=2n)2) Complex lattice…
ex) C-base centered, F = 0 (h+k = Odd)

An example of reciprocal lattice
PDB ID: 1HF4
Egg white lysozyme
Space group: P21
Lattice constant:a = 27.94
b = 62.73
c = 60.25
α = 90.0
β = 90.76
γ = 90.0

Asymmetric unit in reciprocal spacea*Triclinic
1
h k l
±±+

a*

Monoclinic
2/m
±++

b*

c*

b*
a*
b*
a*

Orthorhombic
mmm+++
Tetragonal
4/m
+++

b*
a*
b*
a*
b*
a*
b*
a*

Tetragonal
4/mmm+ + + (h≥k)
Trigonal
3
++±

b*
a*
b*
a*

Hexagonal
6/m+++
Hexagonal
6/mmm+ + + (h≥k)

Trigonal
3m1
+++

Cubic
m3
+ + + (h≥l & k≥l)

Trigonal
31m+ + ± (h≥k)

Cubic
m3m
+ + + (h≥k≥l)X-ray diffraction data collection
Essentials in high quality data collection:
Incident X-ray: Intensity, Divergence, Wavelength
Detector: Detection accuracy, Speed, Image resolution
Crystal: Crystalline order, Size, Radiation resistanceExperimental setup
Laboratory
Rotating anode(Cu, etc)

SpecimeneX-ray mirror
Filament
SORe-Synchrotron

Monochromator

Detector

Metal foil filter(Ni etc)

Collimator,
SlitOscillation method
To record all individual reciprocal spots, the crystal isrotated with a step-width around one axis. The step-width
images are processed to obtain a data set.

ωs0

Rotation angle
Ewald sphere

When a reciprocal
points across theEwald sphere, its
intensity profile isrecorded on detector.

A series of images

Parameters in oscillation method
Detector rotation
Cameradistance

Beamdivergence

1/λ(Ewald sphere)

Crystallographic parameter
Lattice constant, Crystal orientation

Rotation axis

Direct-beam positionDiffraction image processing
Obtain index (hkl) and intensity (I) of each diffraction spot
In collected from single wavelength & single crystal
Software
MOSFLM, XDS (free software)
HKL2000, CrystalClear
Steps of image processing
Indexing:
Determine parameters incl. lattice const.
Integration:
Calculating peak intensity
Scaling:
Merging & averaging equivalent reflections

Spot Finding
Find spots andcalculate and record
its coordinates
on detector.

Autoindexing
Using spot positions, deduce possible crystal system
and lattice parameters.

Lattice type

Lattice constant

Agreement between observed and calculated spot position

Refinement
Various parameters are optimized using spot positions

Refinement

Integration
Integrate diffraction spot profile.
1

I

2

Steps of integration:
1. Estimate correct spot positions
2. Background estimation
3. Fit and integrate by averaged reflection profile

Scaling
Equivalent intensity among symmetrically equivalent reflections

ex. P21; (x, y, z), (x, y+1/2, z)
I(h k l) = I(h k l)I(h k l) = I(h k l)
Estimate scale and falloff factor in each plate
Variation of incident intensity, absorption by crystal, etc.
during one data set
Rmerge overall:
Measures the agreement of symmetry related observations of a reflection.

Rmerge in the last shell:
Rmerge in the highest resolution shell.

I/sigma:
A measure of the signal to noise ratio.

Theory of error ~ 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

Intensity

dependent

Signalnoise

2D detectors for MX
CMOS
Area size(100-400mm)
Resolution(50-100µm)

IP
◎

○

△

◎

○

ME+FOTcm sq. orderby processing tech.

ME

◎

○

◎

○

△

○

Few-200 µm
Phosphor

10 - 100 µm
FOT&phosphor

Few µm100~200 µm~200 µm

50 µm~

◎

○

○

◎

△

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

Noise

Cost

DirectSilicon
Pixel○

Sensitivity

Dynamic
range

Indirect

Amorphous
Selenium

Multi-element

Readout
Speed

Skew

CCD

◎

△

◎

◎

◎

○～◎

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

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)

CCD Detector

CCD detector system

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

Continuous rotation data collection
by CMOS Detector

Hasegawa (JASRI) &Yamamoto (RIKEN)
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

Cryocrystallographic techniquePrevent 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Diffraction power of crystal
Darwin’s Formula4
P ⋅ L ⋅ A ⋅ Vxe2
3
E (h) = λ 2 4⋅ F (h) L
2
ω m c
V

I0

I0: Incident intensity, ω: Angular velocity of crystal rotation, λ: Wavelength,e: Charge of electron, P: Polarization factor (= (1+cos22θ)/2),
L: Lotentz factor (= 1/sinθ 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

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

4

Resolution (Å)

Relationship with B-factor (DWF)

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

6

Resolution limit

Resolution and incident intensity

Incident photon

Reduction by radiation damage

Reflection overlaps
Longer lattice constant gives
narrower spacingof adjacent reflections.
Long axis should be placedalong rotation axis.

Mosaic spread

Spot sharpness depends on crystalline order.

Radiation damage
Bacterial flagelin F41 Crystal@ SPring-8 BL41XU

1st frame

15min.

Very Thin Crystal (≒10µm)

25min.

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

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

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

160

Adiabat150

100 µm thick layer

T(K)40K/Sec
140

100×100×100 µm3 Cubic

130∆T = +20 K

120

100×100×10 µm3 Plate

110∆T = +6.5 K

100
0

1

2

3

4

5

6

7

8

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

Radiation induced formation of reactive radicals (1)
Water

hν

H2 O

H2 O + + e -H2O + + H2O

H+e-

H2 O -+ H2O

H2O - + H2O

H2 O

hν

+ OH

H

+ OH-

H

+ OH

Radiation induced formation of reactive radicals (2)Disulfide bridge(p)-CH2SSCH2-(p)hν(p)-CH2SS + CH2-(p) + e-(p)-CH2SSCH2-(p) + OH(p)-CH2SS + CH2-(p) + OH-(p)-CH2SS + CH2-(p) + OH-(p)-CH2SOH + (p)-CH2S(p)-CH2SSCH2-(p) + OH(p)-CH2SOH + (p)-CH2S

Cysteine(p)-CH2SH + e-(p)-CH2 + SH-(p)-CH2SH + H(p)-CH2 + H2S
Burmeister, Acta Cryst. (2000). D56, 328-341

Radiation induced formation of reactive radicals (3)Aspartate & Glutamatehν(p)-CH2CH2COO(p)-CH2C+ H COO2(p)-CH2C + H2COO- + e(p)-CH2CH2 + CO2

Tyrosinehν(p)-CH2C6H4OH(p)-CH2C6H4OH+(p)-CH2C6H4OH+ + e-(p) -CH2C6H4O + H+

Methionine(p)-CH2CH2SCH3 + 2H(p)-CH2 CH3 + CH3SH
Burmeister, Acta Cryst. (2000). D56, 328-341

Dose limit
Estimated dose limit for ionizing radiation

1.3 × 1017
1 × 1016

keV/mm-3
photon/mm2@12.4keV（Henderson, R. (1990). Proc. R. Soc. London Ser. B, 241, 6-8.）
17
keV/mm-3

4 × 10（Gonzalez, A., Nave, C. (1994). Acta Cryst. D 50, 874-877.）16
photon/mm2@12.4keV

5 × 10（Sliz, P.,Harrison,S.C.Rosenbaum,G., (2002). Structure, 11, 13-19.）

Estimation of radiation damage
SampleSpace group

Hen egg white lysozyme

Lattice

a=b=78.54 ,c=３7.77Å

P4３212α＝β＝γ＝９０°
ＳＲ(ＳＰｒｉｎｇ-８-ＢＬ45ＸＵ)BL
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秒

60秒

Detector

Jupiter210(CCD)ＲＡＸＩＳ-V(ＩＰ)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)

Variation of lattice constants
(Å)

(Å)
36.96

78.80

a,b axes

78.78

36.94
36.92

78.76

a,b

c
78.74

36.90

78.72

36.88

c axis

36.86

78.70

1

Exposure

2

3

4

5

6

7

8

9

10 11 (#data set)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

2.2: Phasing
Crystallographic phase problem
Diffraction intensity is only measureable,
but its phase information is completely lost.
I(hkl) = F(hkl) F*(hkl)
F(hkl) = | F(hkl) | exp iα
Solving methods
1. Direct method
2. Isomorphous replacement (IR)
3. Molecular replacement (MR)

|F| of (a)α of (a)Albert & Lecomte, J. Appl. Cryst. 40, 1153 (2007)

|F| of (b)α of (a)|F| of (a)α of (b)random |F|α of (a)random |Fα of (b)Harker Diagram ~ Structure factor and phase

F(h) =| F(h) | exp iα

Im
i|F(hkl)|= ∑ f j exp 2πih ⋅r j

F(hkl)

Complex spaceαO-|F(hkl)||F(hkl)|Re-i|F(hkl)|
exp iα = cos α + i sin αIsomorphous replacement
Im

FH1

α
O

| FP |: Native| FPH1 | : Derivative 1| FPH2 | : Derivative 2
Re FP = | FP | exp iα

FH2

H1
H2

FPH1 = FP + FH1

Patterson function
Directly calculated from intensity without phase.
The function shows self correlation of electron density.

Interatomic vector u = (u v w)uu-uReal space

Patterson space

In case of few atoms in cell, their coordinates are determined from Patterson function.

Characteristics of Patterson function
1. Even function: P(u) = P(-u)
2. Screw axis in real space > Rotation axis
3. Harker line / Harker section
P21: (x, y, z), (-x, y+1/2, -z)(u, v, w) = (-2x, 1/2, -2z)
4. Correspond to mathematical convolution
f(t)*f(t): Self correlation
f(t)*g(t): Cross corr.

Rough approximation of FH from data
Acentric Zone

Im

| FP |, αP: Native| FPH |, αPH : Derivative| FH |, αH : Heavy atom
FP = | FP | exp iα

α
FH

O

Re

⎧α − α PH ⎫∆F = FPH − FP = FH cos(α PH − α P ) − 2 FP sin 2 ⎨ P⎬
2⎩⎭~ FH cos(α PH − α P )Harker Section
P212121

1: x, y, z, 2: ½-x, -y, ½+z,
3: ½+x, ½-y, -z, 4: -x, ½+y, ½-z

Patterson Peaks p(u,v,w)
2-1: ½-2x, -2y, ½
3-1: ½, ½-2y, -2z
4-1: -2x, ½, ½-2z
3-2: 2x, ½, -2z-½
2-4: ½, -½-2y, 2z
4-3: -½-2x, -2y, ½

2-1: ½-2x, 2y, ½
3-1: ½, ½-2y, 2z
4-1: 2x, ½, ½-2z
3-2: 2x, ½, ½+2z
2-4: ½, ½+2y, 2z
4-3: ½+2x, 2y, ½Relationship among Harker peaks
v
u

u
v=1/2
w

w=1/2

w

v

u=1/2

1: (x, y, z)
2: (½-x, -y, ½+z)
3: (½+x, ½-y, -z)
4: (-x, ½+y, ½-z)

Patterson space (u, v, w)
3-1 (½, ½-2y, -2z), 2-4 (½, -½-2y, 2z)
4-1 (-2x, ½, ½-2z), 3-2 (2x, ½, -½-2z)
2-1 (½-2x, -2y, ½), 4-3 (-½-2x, 2y, ½)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2 Wavelength MAD| F |: Native| Fλ1 (h+) | : Anomalous| Fλ1 (h-) | : Anomalousα| Fλ2 (h+) | : Anomalous| Fλ2 (h-) | : Anomalous
FP=| FP |exp iα

Single phase solution
can be determined.

SAD| F |: Native| Fλ (h+) | : Anomalous| Fλ (h-) | : Anomalousα

Phase probability function shows
bimodal.
>> Phase improvement by density
modification>> High precision data collection

Fall-off factor

Effect of Trichromatic Data Collection
Trichromatic Mode

t

φ=0

1

2 ……

Conventional Modeφ = 0,1,2 ……

0,1,2 ……

0,1,2 ……

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, 1996)

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

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

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

Reference: Park, J.K. et al. J. Bacteriol. 181,6642(1999)

Effect of Trichromatic Data Collection
Data Collection Statistics
Observations

Individuals

I/σRmergeRiso

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

Data
Cho1: Remote

Cho2: Remote

Cho1 (Trichromatic)
Cho2 (Conventional)

Chitosanase Crystal(Length=0.4 µm)

Effect of Trichromatic Data Collection
Variation of Fall-off factor

Cho2(Conventional)

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

Cho2 (Conventional)

Harker section (w = ½)

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

Cho1
RemoteRCullis (iso)#RCullis (ano)0.94

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

8.98

Figure of merit0.6057<|∆φ|>*

Peak

Edge

0.82 /
0.84

0.83 /
0.88

0.91

0.99

8.9 / 14.0

8.1 / 12.5

16.56

7.32

44.2

Remote

0.94

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

0.61671.22 /
0.81

Phasing power#

Cho2(33.9)

1.19 /
0.82
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.

Effect of Trichromatic Data Collection
Phase difference <|φ|> (degree)

Phase difference against true phase65
Cho1(Trichromatic)60

Cho2(Conventinal)

55
50
45
40
35
30
0.03

0.1

0.17

0.24

d-2 (Å -2)

0.31

Effect of Trichromatic Data Collection
Quality of electron density map

Cho1 (Trichromatic)

Cho2 (Conventional)

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

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

Patterson function
Intramolecular・Intermolecular vectors
Real spaceρ(r)= ∫S F(S) exp[-2πi S・r] dS
P(u)= ∫S |F(S)|2 exp[-2πi S・u] dS

Patterson space

Integration radius

Euler angles, α β γ

z’’
x’
z
Rotation axis:⎛cosγ −sinγ 0⎞⎛1 0
0 ⎞⎛cosα −sinα 0⎞
⎜
⎟⎜
⎟⎜
⎟
⎜ sinγ cosγ 0⎟⎜0 cosβ −sinβ⎟⎜ sinα cosα 0⎟
⎜
⎟⎜
⎟⎜
⎟
⎝ 0
0 1⎠⎝0 sinβ cosβ ⎠⎝ 0
0 1⎠
Order
in 3 step rotation: 3
2
1

Polar angles, φψκ

z’’’’

y’’’

z’’
-y’
-z
⎛cosφ −sinφ 0⎞⎛cosϕ 0 −sinϕ⎞⎛cosκ −sinκ 0⎞⎛ cosϕ 0 sinϕ⎞⎛ cosφ sinφ 0⎞
⎟
⎜
⎟⎜
⎟⎜
⎟⎜
⎟⎜
⎜ sinφ cosφ 0⎟⎜ 0 1 0 ⎟⎜sinκ cosκ 0⎟⎜ 0 1 0 ⎟⎜−sinφ cosφ 0⎟
⎟
⎜
⎟⎜
⎟⎜
⎟⎜
⎟⎜
0 1⎠⎝sinϕ 0 cosϕ ⎠⎝ 0
0 1⎠⎝−sinϕ 0 cosϕ⎠⎝ 0
0 1⎠
⎝ 0
5

4

3
Real rotation ~ κ21

An example of rotation function
α

β

γ

ｘ

ｙ

ｚ

Correlation
Coefficient

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

R-factor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=Σ ||Fo|-|Fc(r)||/Σ |Fo|wR2=(Σ w(|Fo|2-|Fc(r)|2)2/Σ w(|Fo|2)2)1/2
Cross validation of R-factor (Rfree)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
Target function

E = Echem + wxray E xray

Exray = ∑h FO (h )− kFC (h )2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 (θ - θide)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
1.5139104.52

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

 9 of 53 beamlines are dedicated to MX

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)

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

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

Industrial Applications of Synchrotron Radiation

Visualization of H-atom

Substrate Complex

Recent topics

Ca2+-ATPase pumps ions
across the sarcoplasmic
reticulum membrane
Chikashi Toyoshima
IMCB, The Univ. of Tokyo
A result of long term proposals

Crystals of 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

cross-sections of an
E1·2Ca2+ crystal

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

25µm

①
②

③
⑥
④
⑤

25µm

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

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

BL control PC

SPring-8

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

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 et al.)New attachment for Hampton/SPINE pins

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

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
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 software

Users lab.

The Internet

SPring-8 OA LAN

SPring-8 Beamline
Access from the out side is restricted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

Control
commandOperation
restrict unit

Device server
(BSS)
BL-WS

Monitoring hutch status
Cannot control devices unlessthe hutch is normally closed.

Robot server
DetectorserverSPring-8 standard protocol
BL interlock
Diffractometer &sample changer

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

F.W.

ffserverffmpegControl
command

Client GUI software

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

DetectorserverDatastorage

Diffractometer &sample changer

Client GUI
software

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

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

0.06nmBL41XU High‐flux Beamline

AtomicAutomation
Automationof
ofStructure
determination
Structure determination

0.12nmHigh‐throughput
BL26B1/2, BL38B1

Amino-Acid

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

0.3nm
Molecule

1nm

100µm

50µm

20µm

112
2008/08/07 山本雅貴

1µm Crystal SizeRIKEN Targeted Proteins beamline BL32XU
for Targeted Proteins Research Program (TPRP)• What is TPRP ?
– Grant: A national project promoted by MEXT, Japan– Aims: To reveal the structure and function of proteins that have great importance inboth academic research and industrial application.
– Research Themes:
Targeted Proteins Research:
Fundamental Biology / Medicine & Pharmacology / Food & Environment
Technology Development:
Protein Production / Structural Analysis / Chemical Regulation / Information
Platform• Beamline Construction– Kunio Hirata, Masaki Yamamoto et al. (RIKEN)

Development of micro-beam beamline
X-ray crystallography of proteins related to human disease and aging.
Micro-beam optimized for Micro-crystal
Standard
Current Limit
Micro-crystal>50µm
20~30µm
<10µm

50µm

50µm

50µm

Target Crystals
・Beam Size
・Flux density
Beam profile of SPring-8 BL41XU

Current
30×30
109Target Beam Size
µ m2
1 ×1
>1010 photons/sec./µm2

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

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

EEM-mirrors for 1 um focusing

Designed mirror surface shape

Kirkpatrick-Baez Mirror
Mirror shape :Elliptical

Mirror length :400 mmEnergy range :8-20 keV

Mirror material :SiO2

Mirror surface :Pt-coated

Glancing angle :3.5mrad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) µm2

20(H) x 19(V) µm2

TC slit size

40(H) x 26(V) µm2

800(H) x 500(V) µm2

Photon flux@12.4 keV

6 x 1010 photons/s

2 x 1013 photons/s

Glancing angle is designed at 3.5mrad

Achieved beam size（2009/11/27）

Intensity / arb. unit

1.0

FWHM
0. 9 µm

0.8
0.6
0.4
0.2
0.0
-3

-2

-1

0

1

Position / um

2

Vertical beam profile
Intensity / arrb. unit

Horizontal beam profile

3

1.0

FWHM
0.9 µm

0.8
0.6
0.4
0.2
0.0
-3

-2

-1

0

1

2

Position / um

Focused photon flux : 6.2x1010 photons/sec
The smallest & highest flux density in the world3Micro-crystal diffractometerHe cryo-cooler

X-ray CCD detector

Air-bearing goniometer
Coaxial microscope

x-ray
He Chamber
Sample changer robot
SPACE

Granite table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 µm/100°-1.5

Spherical steelball

Eccentricity < 0.7µm/360°

-2.0
-2.5
0

（KOHZU PRECISION Co., LTD.）

50

100

150

rotation(d
eg.)
200
250

300

350

400

Tentative diffractometer setting

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

The first crystal onto the 1µm beam

20 µm

Lysozyme

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

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

Protein crystals□

Small molecule crystals

Formula of diffraction power

S = ( F000 / Vcell ) 2 × λ3 × Vcryst
We collect a 2 Å resolution datafrom 2 um lysozyme crystal.

Protein crystal @ BL32XU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.