Beamline 8.3.1 summary

1. Strong PRT and staff

2. Robust optics and endstation

3. Safety: stable, simple operations

4. Funding: Operational funding secure

5. Scientific productivity: high

6. Future: streamlining success

Holton J. M. (2009) J. Synchrotron Rad. 16 133-42

ALS beamline 8.3.1

Diffraction

Methods

Research

Howells et al. (2009) J. Electron. Spectrosc. Relat. Phenom. 170 4-12

1

10

100

1000

1 10 100

e- diffraction - catalase Glaeser 1978

e- tomography - cell Medalia ; Plitzko 2002

e- diff. - purple memb. Hayward 1979

single particle EM Glaeser 2004

predicted Henderson 1990

myrosinase Burmeister 2000

various Silz et al. 2003

bacteriorhodopsin Glaeser et al 2000

ribosome Howells et al 2009

ferritin Owen et al 2006

10 MGy/Aresolution (Å)

max

imu

m t

ole

rab

le d

ose

(M

Gy)

1 2 3 5 7 10 20 40 70 1001

10

100

103

10 MGy/Åwhat the is a MGy?

http://bl831.als.lbl.gov/

damage_rates.pdf

Holton J. M. (2009) J. Synchrotron Rad. 16 133-42

Radiation Damage Model

0.1

0.3

0.5

0.7

0.9

1.1

0 10 20 30 40 50 60 70

Owen et al. (2006) 2CLU * exp(-0.07*dose/d)

accumulated dose (MGy)

no

rmal

ized

to

tal

inte

nsi

ty

Radiation Damage Model

0

10

20

30

40

50

60

70

0 10 20 30 40 50 60 70

lysozyme * exp(-0.07*dose/d) 2CLU * exp(-0.07*dose/d)

accumulated dose (MGy)

bes

t-fi

t B

fac

tor

Kmetko et. al. (2006):

lysozyme: 0.012

apoferritin: 0.017

slopes (Å2/MGy):

lysozyme: 0.013

apoferritin: 0.016

Simulated diffraction imageSimulated diffraction imageMLFSOMMLFSOM

simulatedsimulated realreal

Crystal Size

0

0.1

0.2

0.3

0.4

0.5

0.6

1 10 100 1000

in air

in He

crystal size (μm)

CC

to

co

rrec

t m

od

el

predictedGlaeser et.al. (2000)

1 μm amyloids Nelson et al. 2005 Sawaya et al. 2007

Glaeser et.al. (2000)

Sliz et.al. (2003)

~12 μm xylanase Moukhametzianov et al. 2008

5 μm cypovirus polyhedra Coulibaly et. al. 2007

5 μm (13x) bovine rhodopsin Standfuss et al. 2007theoretical

http://bl831.als.lbl.gov/~jamesh/xtalsize.html

Minimum Crystal Size

nxtal - number of crystals needed

n0 - empirical constant (~ 3)

d - d-spacing of interest (Å)

B - Wilson B factor (Å2)

nxtal = n0

MW VM2

ℓ xℓ yℓ z (d3-1.53) exp(-0.5 B/d2)

MW - molecular weight (kDa)

VM - Matthews number (~2.5 Å3/Da)

ℓ - crystal size (microns)

B ≈ 4 d2 + 12

Holton J. M. (2009) J. Synchrotron Rad. 16 133-42

http://bl831.als.lbl.gov/~jamesh/xtalsize.html

Where:IDL - average damage-limited intensity (photons/hkl) at a given resolution

105 - converting R from μm to m, re from m to Å, ρ from g/cm3 to kg/m3 and MGy to Gy

re - classical electron radius (2.818 x 10-15 m/electron)

h - Planck’s constant (6.626 x 10-34 J∙s)c - speed of light (299792458 m/s)fdecayed - fractional progress toward completely faded spots at end of data set

ρ - density of crystal (~1.2 g/cm3)R - radius of the spherical crystal (μm)λ - X-ray wavelength (Å)fNH - the Nave & Hill (2005) dose capture fraction (1 for large crystals)

nASU - number of proteins in the asymmetric unit

Mr - molecular weight of the protein (Daltons or g/mol)

VM - Matthews’s coefficient (~2.4 Å3/Dalton)

H - Howells’s criterion (10 MGy/Å)θ - Bragg anglea

2 - number-averaged squared structure factor per protein atom (electron2)

Ma - number-averaged atomic weight of a protein atom (~7.1 Daltons)

B - average (Wilson) temperature factor (Å2)μ - attenuation coefficient of sphere material (m-1)μen - mass energy-absorption coefficient of sphere material (m-1)

Theoretical limit:

Holton J. M. and Frankel K. A. (2010) Acta D submitted

22

sphere

2

4425 sin2exp

sin

4cos3

01

)2(T

sin2ln

5.0

f

f10

9

2B

M

f

θ

θ

,R,μT

θ ,μ,R λH

VMn

λρR

hc

rI

a

a

ensphereMrASUNH

decayedeDL

Theoretical limit:

at ~2.4 Å

photon

spot μm3 1.0

Holton J. M. and Frankel K. A. (2010) Acta D submitted

for lysozyme

Optimum exposure time(faint spots)

2

00 10

gain

mt

t

gain

bgbg

ref

hrref

thr optimum exposure time for data set (s)tref exposure time of reference image (s)bgref background level near weak spots on

reference image (ADU)bg0 ADC offset of detector (ADU)σ0 rms read-out noise (ADU)gain ADU/photonm multiplicity of data set (including partials)

Short answer:

bghr = 90 ADU

for ADSC Q315r

Specific Damage

Damage changes absorption spectrum

0

500

1000

1500

2000

2500

3000

3500

4000

4500

50001

26

40

12

64

5

12

65

0

12

65

5

12

66

0

12

66

5

12

67

0

12

67

5

12

68

0

12

68

5

12

69

0

12

69

5

12

70

0

beforebeforeburntburnt

Photon energy (eV)

coun

ts

1

0

Holton J. M. (2007) J. Synchrotron Rad. 14 51-72

fluorescence probe for damage

fluence (1015 photons/mm2)

Fra

ctio

n u

nco

nve

rted

25mM SeMet in 25% glycerol

0.

0

0

.2

0.4

0.6

0

.8

1.0

0 50 100 150 200 250 300 350 400

Exposing at 12680 eV

Se cross-section at 12680 eV

Holton J. M. (2007) J. Synchrotron Rad. 14 51-72

fluorescence probe for damage

Absorbed Dose (MGy)

Fra

ctio

n u

nco

nve

rted

Wide range of decay rates seen

0.

0

0

.2

0.4

0.6

0

.8

1.0

0 50 100 150 200

Half-dose = 41.7 ± 4 MGy“GCN4” in crystal

Half-dose = 5.5 ± 0.6 MGy8 mM SeMet in NaOH

Protection factor: 660% ± 94%

Holton J. M. (2007) J. Synchrotron Rad. 14 51-72

Protective factors for SeMet

0

50

100

150

200

not i

ceno

t nan

oice

low

pH

asco

rbat

eni

trate

low

tem

pera

ture

in p

eptid

efo

lded

crys

talliz

edG

CN4

xta

lno

t NE1

xta

lic

e vs

GCN

4

protective measure

pro

tec

tio

n f

ac

tor

(%)

750

%

Holton J. M. (2007) J. Synchrotron Rad. 14 51-72

Take-home lesson:

radiation damage to metal sites is unpredictable

Best strategy:

5 MGy to complete data

geometrically increasing exposure

Holton J. M. (2007) J. Synchrotron Rad. 14 51-72

Minimum required signal (MAD/SAD)

"#

)(3.1

fsites

DaMW

sd

I

dataset 1 2-11 12

exposure 1.0s 0.1s 1.0s

frames 100 100 x 10 100

Rmerge 5.6% 11.2% 4.7% Ranom 4.8% 4.7% 4.7%

I/sd 29.5 43.4 33.3 I/sd (2.0 Ǻ) 23.3 29.6 25.8

redundancy 7.6 75.7 7.6

PADFPH 36.69 37.11 37.93

FOM 0.342 0.343 0.366

FOMDM 0.698 0.711 0.726

CC(1H87) 0.418 0.492 0.468

same total dose with high and low redundancy

Spatial Noise

down up

Rseparate

Spatial Noise

odd even

Rmixed

Spatial Noise

separate:

mixed:

2.5%

0.9%

2.5%2-0.9%2 = 2.3%2

Spatial Noise

mult > (—)22.3%

<ΔF/F>

Minimum Crystal Size

nxtal - number of crystals needed

n0 - 3 for complete data set, 180 for MAD

d - d-spacing of interest (Å)

B - Wilson B factor (Å2)

nxtal = n0

MW VM2

ℓ xℓ yℓ z (d3-1.53) exp(-0.5 B/d2)

MW - molecular weight (kDa)

VM - Matthews number (~2.5 Å3/Da)

ℓ - crystal size (microns)

B ≈ 4 d2 + 12

Holton J. M. (2009) J. Synchrotron Rad. 16 133-42

http://bl831.als.lbl.gov/~jamesh/xtalsize.html

Take-home lesson:

need better crystals for MAD

Best strategy:

find them

accurate, unattended

data colleciton

beamlinemicroscope

referenceimage

Re-centering

accurate, unattended

screening

sample shadow on detector

Cu

sample shadow on detector

X-ray shadow of cryo stream

Plate goniometers