douglas instruments microseeding slide 1 microseeding matrix screening and other developments in...
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Microseeding slide 1
Microseeding Matrix Screening and other developments in automatic protein crystallization
Patrick Shaw Stewart
Douglas Instruments Limited (near Oxford, UK):Peter Baldock, Patrick Shaw Stewart, James Smith
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Microseeding slide 2
Microseeding in screening experiments
Allan D’ArcyNovartis, Basle
2006 ‘Matrix-seeding script’
An automated microseed matrix-screening methodfor protein crystallization. Acta Cryst. (2007). D63, 550–554
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Microseeding slide 3
Phase diagram of a protein
[Protein]
[Precipitant]
clear
precipitate
nucleation
metastable zone
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Microseeding slide 4
Microseeding in screening experiments
Gregory Ireton and Barry Stoddard.
'Microseed matrix screening to improve crystals of yeast cytosine deaminase'. Acta Crystallographica section D60
(2004), 601–605.
With Ca2+ conditions they couldn’t get crystals without microseeding.
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Microseeding slide 5
Phase diagram of a protein
[Protein]
[Precipitant]
clear
precipitate
metastable zone
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Microseeding slide 6
Microseeding in screening experiments
1. May explain why there are SO many hits with MMS
2. Can be used to CONTROL number of conditions
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Microseeding slide 7
Microseeding in screening experimentsJames Smith 2006 ‘Matrix-seeding script’
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Microseeding slide 8
Microseeding in screening experimentsJames Smith 2006 ‘Matrix-seeding script’
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Microseeding slide 9
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 wells
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Microseeding slide 10
Microseeding in screening experiments
No seeds
Laura CendronUniversity of Padova
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Microseeding slide 11
Microseeding in screening experiments
No seeds Seeds
Laura CendronUniversity of Padova
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Microseeding slide 12
Phase diagram of a protein
[Protein]
[Seeds]
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Microseeding slide 13
Mini-optimization
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Microseeding slide 14
Under £24,000Under $46,000
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Microseeding slide 15
Bullet points:
• 100 + 100 nl sitting drop
• Only 9.8 μl of protein to set up a 96-well plate
• Additive screens
• Microseeding
• Mini-optimization and optimization
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Microseeding slide 16
Jonathan Hadden
• 96-well plate takes 8 minutes
• There is an evaporation shield
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Microseeding slide 17
Under £24,000Under $46,000
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Microseeding slide 18
ORYX (Arabian)
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