Optimizing structure determination
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Optimizing structure determination
How many are we solving?
What is the limit?
Are we there yet?
Why not?
What are the biggest problems?
How many are we solving?http://asdp.bnl.gov/asda/Libraries/pdb_statis/latest/bml/ALS.html
$$ → photons
photons → data
data → models
models → results
results → $$
Breaking it down
Seconds Description Percent
104490 Assigned and available 91%
42093 Shutter open 40%
52684 Collecting (3026 images) 50%
51806 Something else 50%
Operational Efficiency“representative” 8.3.1 user
Seconds Description Percent
51806 Something else 100%
247s 45 Mounting 22%
229s 37 Centering 16%
179s 109
Strategizing 38%
309s 37 Prepping 24%
Operational Efficiency“representative” 8.3.1 user
Number Description Percent
446028 Images (~7 TB) 33%
2346 Data sets 47%
449 MAD/SAD (1:2) 19%
48 Published 2%
8.3.1 in 2003
Turning data into models
Top producing beamlines of the world
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(all 5)
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http://asdp.bnl.gov/asda/Libraries/pdb_statis/latest/bml/ALL.html
Str
uct
ure
s cr
edit
ed
28 operating US beamlines
~1011 ph/μm2 exposure limit
÷ 2x109 ph/μm2/s
~ 100,000 datasets/year
÷ 1324 str in 2003
~ 2% efficient
What is the limit?
DVD data archive
Elven Automation
Elves examine images andset-up data processing
Elves run…
mosflmscalasolve
mlpharedm
arp/warp
Apr 6 – 24 at ALS 8.3.1
Elven Automation
27,686 images collected
148 datasets (15 MAD)
31 investigators
56 unique cells
5 KDa – 23 MDa asymmetric unit
0.94 – 32 Å resolution (3.2 Å)
Apr 6 – 24 at ALS 8.3.1
Elven Automation
148 datasets
117 succeded
~3.5 (0.1-75) hours
31 failed
~61 (0-231) hours
2 / 15 MAD structures
Overlaps
Signal to noise
Radiation Damage
Why do structures fail?
avoidable overlaps
mosaicity
phi
dete
ctor
c*
b
c
a
Ewald sphere
unavoidable overlaps
mosaicity
phi
dete
ctor
c*
b
c
a
Ewald sphere
Overlaps
Signal to noise
Radiation Damage
Why do structures fail?
MAD phasing simulation
-0.2
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0.2
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0.8
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0.01 0.1 1 10
Anomalous signal to noise ratio
Cor
rela
tion
coef
ficie
nt t
o co
rrec
t m
odel
mlphare results
SAD phasing simulation
-0.2
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0.01 0.1 1 10
Anomalous signal to noise ratio
Cor
rela
tion
coef
ficie
nt t
o co
rrec
t m
odel
mlphare results
Minimum required signal (MAD/SAD)
"#
)(3.1
fsitesDaMW
sd
I
Is it Is it realreal, or is it , or is it MLFSOMMLFSOM??
“We really need those
high-resolution spots”
Incremental strategy
incremental_strategy.com merged.mtz auto.mat
Overlaps
Signal to noise
Radiation Damage
Why do structures fail?
Distention of cryo with dose
before
Distention of cryo with dose
after
Water ring shiftsaturated sucrose in 250mM WO4
0 MGy
Water ring shiftsaturated sucrose in 250mM WO4
184 MGy
Water ring shift
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100
0.00 0.07 0.13 0.20 0.27
0 MGy
37 MGy80 MGy
187 MGy
Resolution (Ǻ)
Ph
oto
ns/
s/p
ixel
7.5 3.8 2.5 1.9 1.5
saturated sucrose in 250mM WO4
Protein crystal background
Water ring shift
3.678
3.679
3.68
3.681
3.682
3.683
3.684
3.685
3.686
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Absorbed dose (MGy)
Wat
er r
ing
posi
tion
(Ǻ)
GCN4-p1-N16A trigonal crystal
3.555
3.565
3.575
3.585
3.595
3.605
3.615
crystal backgroundsaturated sucrose
Water ring shift
http://www.lsbu.ac.uk/water/amorph.html
Water ring shiftbubbles?
Richard D. Leapman, Songquan Sun, Ultramicroscopy (1995)
Water ring shiftHydrogen bubbles?
Richard D. Leapman, Songquan Sun, Ultramicroscopy (1995)
Water ring shiftHydrogen bubbles?
http://www.rcdc.nd.edu/compilations/Rxn.pdf
“The hydrogen atom reacts with organic compounds by abstracting H from saturated molecules and by adding to centers of unsaturation,
for example,
Damage model system
Data quality vs phasing quality
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model vs warpwarp vs experiment
Exposure time (min)
Cor
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coef
ficie
nt
Individual atoms decay at different rates
00.10.20.30.40.50.60.70.80.9
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all atoms
Se #1
Se #5
Exposure time (min)
Cor
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coef
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o ob
serv
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ata
Damage changes fluorescence spectrum
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beforebeforeburntburnt
Photon energy (eV)
coun
ts
Damage changes fluorescence spectrum
fluence (103 photons/mm2)
Fra
ctio
n u
nco
nve
rted
25mM SeMet in 25% glycerol
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Exposing at 12680 eV
Se cross-section at 12680 eV
fluorescence probe for damage
Absorbed Dose (MGy)
Fra
ctio
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nco
nve
rted
Wide range of decay rates seen
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Half-dose = 41.7 ± 4 MGy“GCN4” in crystal
Half-dose = 5.5 ± 0.6 MGy8 mM SeMet in NaOH
Protection factor: 660% ± 94%
“Can we do more
with what we’ve got?”
Interleaved Schedulingexperiment queue beamline
Minor 30s
Choe 120s
Alberta 60s
Alberta 60s
Choe 30s
Minor 30s
SuperTongSuper Tong
SuperTong
“infinite capacity” sample carousel
Carousel open
CHL idlepos
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