A4

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Microseed it! Patrick Shaw Stewart Douglas
Instruments Limited (near Oxford, UK) Peter
Baldock, Patrick Shaw Stewart, Richard Briggs,
James Smith
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• 100 100 nl sitting drops
• Up to 2 2 µl sitting drops!
• Only 9.8 µl of protein to set up a 96-well plate
• Microseeding (MMS)
• Mini-optimization (all systems) and
• Additive screens (Oryx4 and Oryx8)
• Microbatch-under-oil (Oryx4 and Oryx8)
• Multi-dimensional optimization (Oryx8)
• Dispensing reservoirs (Oryx8 - in future)

3

• Bullet points
• 100 100 nl sitting drops
• Up to 2 2 µl sitting drops!
• Only 9.8 µl of protein to set up a 96-well plate
• Microseeding (all systems)
• Additive screens (Oryx4 and Oryx8)
• Mini-optimization (all systems) and optimization
  (Oryx8)

4

• MMS microseeding
• Scaling up from nanodrops

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Seeding

• Macroseeding
• Microseeding
• Streak seeding
• De Titta seed bead
• MMS microseeding

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MMS microseeding

• Seeding into random SCREENS
• More hits
• Better crystals

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Microseeding in screening experiments
Allan DArcy Novartis, Basle 2006
Matrix-seeding script
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Microseeding in screening experiments
Allan DArcy Novartis, Basle 2006 Matrix-seeding
script
3-bore tip
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Microseeding in screening experiments
Allan DArcy Novartis, Basle 2006 Matrix-seeding
script
3-bore tip
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Microseeding in screening experiments
Allan DArcy, Novartis, Basle. 2006
Matrix-seeding script
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Microseeding in screening experiments
Allan DArcy Novartis, Basle 2006
Matrix-seeding script An automated microseed
matrix-screening method for protein
crystallization. Acta Cryst. (2007). D63,
550554
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Microseeding in screening experiments
DArcy et al. Acta Cryst. (2007). D63
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Microseeding in screening experiments
DArcy et al. Acta Cryst. (2007). D63
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Microseeding in screening experiments
DArcy et al. Acta Cryst. (2007). D63
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Microseeding in screening experiments
USP7 crystals used for seeds grown in 30 PEG
3350, 100 mM HEPES pH 7.0
USP7 crystals after seeding in 20 PEG 3350, 200
mM magnesium hexahydrate
DArcy et al. Acta Cryst. (2007). D63
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• Seed stock is made up in the reservoir solution
  where the hit was found (e.g. Nextal 12)
• Break crystals
• Place contents of well in 50 µl of reservoir
  solution
• Vortex with Hampton Seed Bead
• Make a dilution series immediately
• Freeze!

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Phase diagram of a protein
precipitate
nucleation
Protein
metastable zone
clear
Precipitant
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Microseeding in screening experiments
Gregory Ireton and Barry Stoddard.
'Microseed matrix screening to improve crystals
of yeast cytosine deaminase'. Acta
Crystallographica section D60 (2004), 601605.
With Ca2 conditions they couldnt get crystals
without microseeding.
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Phase diagram of a protein
precipitate
Protein
metastable zone
clear
Precipitant
20
Microseeding in screening experiments
DArcy et al. Acta Cryst. (2007). D63
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Microseeding in screening experiments

• Lesley Haire, NIMR
• 6 hits in a screen, all poorly formed.
• Seeding - 30 hits, including several well-formed
  crystals.
• Jens-Christian Navaro Poulsen, University of
  Copenhagen
• 1 hit in 288 wells
• Seeding - 10 hits in 96 wells
• Using one of those crystals for a
  second-generation seeding experiment - another 10
  hits in 96 different conditions

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Microseeding in screening experiments
No seeds
Seeds

Laura Cendron University of Padova
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Microseeding in screening experiments
Jens-Christian Navaro Poulsen, University of
Copenhagen Use control to identify salt crystals
0.3 µl protein 0.25 µl reservoir 0.05 µl seeds
- 0.25 µl reservoir 0.05 µl seeds
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Identifying protein / salt crystals

• UV LED

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Identifying protein / salt crystals

• UV LED

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Identifying protein / salt crystals

• Label with ANS
• Intrinsic fluorescence

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Microseeding in screening experiments
Explanation 1 the seeds 50 of extra
hits Explanation 2 the solution containing
seeds 50 of extra hits Preliminary results
at Douglas Instruments show 5050
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Microseeding in screening experiments

• Explanation 1 the seeds 50 of extra hits
• Explanation 2 the solution containing seeds
  50 of extra hits
• Preliminary results at Douglas Instruments show
  5050
• St John et al (Edwin Pozharski lab.) Acta Cryst.
  (2008). D64, 12221227
• 75 (solution) 25 (seeds)
• Diluted seed stock 1/100
• Data mainly from model proteins

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Scaling up
1 1 µl
100 100 nl
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Scaling up
1 1 µl PRECIPITATION !!
100 100 nl
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Scaling up
Low surface to volume ratio
High surface to volume ratio

• More protein is lost at the air/liquid interface
• Equilibration is faster

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Scaling up
Scales up to 1 1 µl (Heather Ringrose, Pfizer)
Try 200 nl (protein) 100 nl (reservoir
solution)
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Scaling up
Scales up to 1 1 µl (Heather Ringrose,
Pfizer) Increase the salt by 50 100
Try 200 nl (protein) 100 nl (reservoir
solution) Equilibrates faster
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MMS comments by Allan DArcy

• Freeze your seed stock then you can always
  reproduce your crystals (even years later)

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MMS comments by Allan DArcy

• Freeze your seed stock then you can always
  reproduce your crystals (even years later)
• Usually you dont need to optimize after MMS

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MMS comments by Allan DArcy

• Freeze your seed stock then you can always
  reproduce your crystals (even years later)
• Usually you dont need to optimize after MMS
• So always do it unless you solve the structure
  by harvesting crystals straight from your screens