Origin of a Species: History and observations of one high throughput crystallization laboratory J....
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Transcript of Origin of a Species: History and observations of one high throughput crystallization laboratory J....
Origin of a Species: History and observations of one high throughput
crystallization laboratory
J. R. Luft, R. J. Collins, S. M. Gulde, A.M. Lauricella, C. A. Mancuso, J. L. Smith,
C. K. Veatch, and G. T. DeTitta
Recent Advances in Macromolecular CrystallizationLe Bischenberg, France
May 8-11, 2005
Hypothesis
By executing a large number of precipitation reactions for a large number of proteins we could predict crystallization conditions for a previously un-crystallized protein by comparison of precipitation behavior.
Protein Binary Code PSSUnknown 1011010010110010 --
Protein B 0111010111010010 11Protein A 0001100101010101 5
What is the most efficient way to execute precipitation reactions?
• Batch, in particular microbatch in a capillary– Minimize solution volume requirements for
precipitation cocktails
Good, but not Great• Maximum throughput (manual)
50 experiments/technician/day• Crystals were a ‘by-product’.
Microbatch Under Oil
Chayen, N.E., Stewart, P.D.S., Blow, D.M., Journal of Crystal Growth, 122 (1992) 176-180.
• Minimal solution volumes
• Plates for storage and handling
Mothers and Daughters
• Mother (source) / Daughter (destination) plates• Efficient parallel setups
…
source destination
Solution Delivery
• Pump
• Slot pins
• Pipettes
• Syringes
• Faster Syringes
http://www.vp-scientific.com/index.htm
Screening for Crystallization Leads
* 15361. Oil2. Crystallization Cocktail3. Macromolecular Solution
Quantity• How Many Experiments?
– 20 plates per day (~ 200/month)– 200(plates) x 1536(experiments) x 6(images)
• Generates 1.8 million images/month
As of 3/17/2005:4877 (plates) x 1536 (experiments)
7.5 million experiments
45 million images
469 active users
1. The Plate
• February 2004– Square to circular cross-section well
• Better visibility
• Now in production at Greiner BioOne– LBR plates
• “significantly higher water absorption”
11
22
2. Cocktail Dehydration
• Plates prepared with oil
• Cocktail added to plates (200nl)
• Plates are stored at 4oC for 1 day – 2 weeks
[M]
[C]
Dehydrate C drop
Decrease Vc drop
Increase [C]Decrease dilution of [M]Increase [M]exp
??
3. The Oil
• Paraffin Oil – (IR Spec grad)
• Emulsion formed with some batches
• Mineral Oil – (USP grade)
• December 2004
4. [Salt] in the Samples
• Compared salt concentrations for 392 samples– ([KCl] + [NaCl]) mM
• All samples were set up using the ‘standard’ 1536 screen and outcomes reviewed
• Compared 158 samples shipped:
– 76 Frozen (dry ice)– 82 Liquid (wet ice)
• All samples were set up using the ‘standard’ 1536 screen and outcomes reviewed
5. State of the Samples
Frozen Samples versus Liquid Samples
frozenliquid
Samples with hits Samples with no hits
Num
ber
of S
ampl
es
• Work supported in part by the John R. Oishei Foundation, the Cummings Foundation
• NIH RR016924, NIH P50 GM-62413 and NIH P50 GM-64655• Special thanks to Bob Cudney• Greiner Bio-One
• And ….
Acknowledgements
http://www.hwi.buffalo.edu