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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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

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mlphare results

SAD phasing simulation

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mlphare results

Minimum required signal (MAD/SAD)

"#

)(3.1

fsitesDaMW

sd

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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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37 MGy80 MGy

187 MGy

Resolution (Ǻ)

Ph

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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

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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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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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Damage changes fluorescence spectrum

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beforebeforeburntburnt

Photon energy (eV)

coun

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Damage changes fluorescence spectrum

fluence (103 photons/mm2)

Fra

ctio

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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

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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

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Choe 120s

Alberta 60s

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