beamline 8.3.1 summary 1.strong prt and staff 2.robust optics and endstation 3.safety: stable,...
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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
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