Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why...
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![Page 1: Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why not? What are the biggest problems?](https://reader036.fdocuments.us/reader036/viewer/2022062518/56649f3c5503460f94c5af5a/html5/thumbnails/1.jpg)
Optimizing structure determination
How many are we solving?
What is the limit?
Are we there yet?
Why not?
What are the biggest problems?
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How many are we solving?http://asdp.bnl.gov/asda/Libraries/pdb_statis/latest/bml/ALS.html
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$$ → photons
photons → data
data → models
models → results
results → $$
Breaking it down
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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
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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
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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
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Top producing beamlines of the world
0
20
40
60
80
100
120
140
160
180
AP
S 19ID
AL
S 8.3.1
ES
RF
ID14-1
ES
RF
ID14-4
PF
(all 5)
ES
RF
ID14-2
NS
LS
X4A
AP
S 22ID
ES
RF
ID29
AP
S 19B
M
CH
ES
S (all 4)
AL
S 8.2.1
AL
S 5.0.2
HA
SY
LA
B X
11
HA
SY
LA
B B
W6
AL
S 8.2.2
SL
S (b
oth
)
ES
RF
BM
14
SP
RIN
G8 26B
1
AP
S 14B
MC
NS
LS
X25
AL
S 5.0.1
SS
RL
BL
9-1
NS
LS
X12C
2002
2004
http://asdp.bnl.gov/asda/Libraries/pdb_statis/latest/bml/ALL.html
Str
uct
ure
s cr
edit
ed
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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?
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DVD data archive
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Elven Automation
Elves examine images andset-up data processing
Elves run…
mosflmscalasolve
mlpharedm
arp/warp
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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)
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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
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Overlaps
Signal to noise
Radiation Damage
Why do structures fail?
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avoidable overlaps
mosaicity
phi
dete
ctor
c*
b
c
a
Ewald sphere
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unavoidable overlaps
mosaicity
phi
dete
ctor
c*
b
c
a
Ewald sphere
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Overlaps
Signal to noise
Radiation Damage
Why do structures fail?
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MAD phasing simulation
-0.2
0
0.2
0.4
0.6
0.8
1
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
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SAD phasing simulation
-0.2
0
0.2
0.4
0.6
0.8
1
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
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Minimum required signal (MAD/SAD)
"#
)(3.1
fsitesDaMW
sd
I
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Is it Is it realreal, or is it , or is it MLFSOMMLFSOM??
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“We really need those
high-resolution spots”
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Incremental strategy
incremental_strategy.com merged.mtz auto.mat
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Overlaps
Signal to noise
Radiation Damage
Why do structures fail?
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Distention of cryo with dose
before
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Distention of cryo with dose
after
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Water ring shiftsaturated sucrose in 250mM WO4
0 MGy
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Water ring shiftsaturated sucrose in 250mM WO4
184 MGy
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Water ring shift
40
50
60
70
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90
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
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Protein crystal background
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Water ring shift
3.678
3.679
3.68
3.681
3.682
3.683
3.684
3.685
3.686
0 10 20 30 40 50 60
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
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Water ring shift
http://www.lsbu.ac.uk/water/amorph.html
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Water ring shiftbubbles?
Richard D. Leapman, Songquan Sun, Ultramicroscopy (1995)
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Water ring shiftHydrogen bubbles?
Richard D. Leapman, Songquan Sun, Ultramicroscopy (1995)
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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,
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Damage model system
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Data quality vs phasing quality
0
0.1
0.2
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0.7
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0 20 40 60 80 100
model vs warpwarp vs experiment
Exposure time (min)
Cor
rela
tion
coef
ficie
nt
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Individual atoms decay at different rates
00.10.20.30.40.50.60.70.80.9
1
0 20 40 60 80 100
all atoms
Se #1
Se #5
Exposure time (min)
Cor
rela
tion
coef
ficie
nt t
o ob
serv
ed d
ata
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Damage changes fluorescence spectrum
0
500
1000
1500
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50001
26
40
12
64
5
12
65
0
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65
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66
5
12
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0
12
67
5
12
68
0
12
68
5
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69
0
12
69
5
12
70
0
beforebeforeburntburnt
Photon energy (eV)
coun
ts
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Damage changes fluorescence spectrum
fluence (103 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 20 40 60 80 100 120
Exposing at 12680 eV
Se cross-section at 12680 eV
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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%
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“Can we do more
with what we’ve got?”
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Interleaved Schedulingexperiment queue beamline
Minor 30s
Choe 120s
Alberta 60s
Alberta 60s
Choe 30s
Minor 30s
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SuperTongSuper Tong
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SuperTong
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“infinite capacity” sample carousel
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Carousel open
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CHL idlepos