Weizmann microbatch

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Microbatch crystallisation for screening worth
a try?
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Microbatch crystallisation

• Description of the method
• Modified microbatch and the influence of
  different oils
• Modified microbatch compared to vapour diffusion
• Advantages and disadvantages

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Batch Crystallisation

• Batch method
• Where the conditions to produce crystals are
  pre-determined, no concentration of the solution
  is required large drops may be used or even on an
  industrial scale.
• Probably the case in most vapour diffusion
  experiments

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Microbatch Crystallisation
Microbatch method Using small volumes of
protein(1ul) and precipitating agent under a
layer of paraffin oil (Chayen et. al. 1990) Used
for optimization not screening
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Microbatch Crystallisation
Automated microbatch Douglas Instruments
develops a robot for setting up crystallisation
drops under paraffin oil.(1992)
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Microbatch Crystallisation the early days
In 1994 Roche decides to use the Douglas system
for crystallisation screens Far fewer hits were
obtained than with standard hanging drop vapour
diffusion
If you buy a robot use it!!
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Microbatch Crystallisation the early days
Different oils were tested and a correlation was
found between viscosity of the oil and the rate
of crystals appearance
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Als Oil is born!!
Modified Microbatch or microbatch diffusion By
modifying the oils used a system is established
which allows concentration of the protein and
precipitating agent in the drop.
A novel approach to crystallising proteins under
oil A.DArcy, C. Elmore, M. Stihle, J.E.
Johnston Journal of Crystal Growth 1996 168
175-180
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The crystallisation experiment
Many drops remain clear
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Microbatch reaches higher concentrations
Water diffuses through the oil, the rate is
affected by the type of the oil
6ml
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Manual microbatch
A
B
C
D
Manual microbatch setup 1. Oil pipetted into
wells of 96 well plates (Figs. 4A, 4B) 2.
Reagents pipetted using 8 channel pipette 3.
Samples pipetted using single channel pipette 4.
Plates centrifuged (5 minutes, 2500 rpm) to
coalesce drops 5. Plates sealed
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Manual microbatch
As many as 40 screens or 4000 drops can be set
up in an eight hour working day. This is well
above the number of screens a normal
(non-structural genomics) lab would need to run.
However setting up a standard 96 condition screen
by hand in approximately 10 minutes is quite
attractive considering the low cost of the
equipment needed (centrifuge, and pipettes).
(R. Cudney) personal communication
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Automated microbatch
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Douglas Impax system used from 1994- 2000 at La
Roche 2000-2003 at Morphochem
Twin tube for protein ppt.agent
72 well plate
Screen solutions
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Oryx system in use at the protease platform
Novartis since 2004

• We can finally do both methods

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It makes screening simple
Microbatch
Vapour diffusion

• Reservoir solutions made every time
• Drops may dry out during pipetting
• More protein may be needed with some systems
  (Cartesian)
• Reservoirs and drops pipetted in 2 steps
• Plates must be sealed

• Reservoir solutions made once per 100-200
  experiments
• No drying out, drops covered with oil
• Very little extra protein required
• No reservoirs need to be pipetted
• No sealing required

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Other Robotic Sytems Used For Microbatch
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Different plates

• HLA Vapour batch Imp_at_ct

• Good drops, oil well contained, reservoir
  included.
• Poor optics with black plate

Good drops but messy

• Good drops, oil well contained, reservoir
  included.
• Skin formation and faster drying

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Pipetting modes Standard and Douglas
Protein is aspirated and dispensed from tube 1
Standard pipetting mode
each crystallisation reagent is aspirated and
then dispensed with protein
Protein and precipitating agent are pipetted
together
There seems to be more hits with the
simultaneous pipetting
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Oil first or last??

• It is possible to pipette the protein and
  crystallising agent under a layer of pre-poured
  oil
• protein may not drop off tubing, static can be a
  problem, may need to grease tubing, de-charge
  plates Source plate) and investigator!!
• It is also possible to pipette in a vapour
  diffusion mode and add oil afterwards
• now preferred mode

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The effect of different oils in microbatch
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Different oils give different results

• Glucose isomerase after 4 days using a 48
  condition screen in microbatch
• Paraffin oil 4 hits
• Als oil (Silicone/Paraffin) 13 hits
• Silicone oil 28 hits

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The importance of the type of oil
Twice as many hits with silicone oil
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The problem with silicone oil
Condition 1
Silicone/Paraffin
Silicone
Day 1
Day 1
The drops dry out
Day 4
Day 4
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6 Days with and without reservoir
Silicone oil with reservoir
Silicone oil without reservoir
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Stabilising crystals grown under silicone oil
Average number of hits with 4 different proteins
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The oil is more important than the size of the
screen
In a recent study for a structural genomics
program paraffin oil was used in the microbatch
system to set up 1536 conditions

• Samples suitable for crystallization 22
• Success rate (on suitable samples) 36
• Our success rate is 70-80 with 48 solutions

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Other oils used

• Juvaniyama et al. Characterization,
  crystallization and preliminary Xray analysis of
  bifunctional dihydrofolate reductase-thymidylate
  synthase from Plasmodium falciparum
• Used Cussons Baby oil as a substitute for Als
  Oil for its lower volatility, purity, and
  availability. The oil is a mixture of mineral
  oil, olive oil, and vitamin E, the presence of
  vitamin E might also help as oxygen scavenger
• Composition of other baby oils paraffin oil,
  Triticium vulgare, (wheat) Prunus
  dulcis,(almonds) Perfume Isopropanol, palmate

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Small volumes dont mean small crystals
200200nl drops, aldolase with crystals at low
and high magnification (100-200um)
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Microbatch and temperature
Vapour diffusion experiments are not suitable for
temperature gradients because condensation tends
to form on the inside of the coverslide.
Drop volume will increase
Increased temperature
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Microbatch and temperature
This is not the case with microbatch at higher
temperatures the evaporation and concentration
will be accelerated
Temperature gradient experiments can be used as
an additional variable in crystallisation
screening or as an optimisation tool.
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Using temperature gradients with microbatch
Crystallisation 48 crystallisation conditions was
set up at 3 temperatures. The screens were scored
after for a period of 5 days.
4 20 Gradient
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Word of caution

• Be careful with microbatch crystallisation which
  have been setup at 4 deg but need to be observed
  at room temp.
• Do not remove lid!!!

• Vapour from the air will condense on the oil and
  eventually mix with the drops

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Microbatch and vapour diffusionis there really
a difference?
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Comparison of modified microbatch and vapour
diffusion

• Same experiment in microbatch and vapour
• diffusion drops observed on setup

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Examples of two proteins that require microbatch
crystallization
Comparison of modified microbatch and vapour
diffusion
Microbatch
Vapour Diffusion
Identical solutions used in both cases
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Screening studies comparing microbatch with
vapor diffusion

P.F.M. Baldock, V. Mills, P.D. Shaw Stewart. A
comparison of microbatch and vapour diffusion for
initial screening of crystallization conditions.
Journal of Crystal Growth. 168 (1996), pp 170-174
or http//www.douglas.co.uk/rep2.htm A. DArcy,
G.E. Dale, M. Stihle, B. DArcy. Results
reported at the 8th International Conference on
the Crystallization of Biological Macromolecules,
May 18, 2000. N. Noordeen and S. Cowan-Jacob.
Novartis Pharma AG. http//www.hamptonresearch.com
/stuff/ppt_files/P6.ppt Misuaki Sugahara, Riken
Harima Institute, SPring8. Personal
communication.
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Experiences with the use of microbatch under oil
method of crystallisation Nafeesa Noordeen and
Sandra Cowen-Jacob Novartis Basel
Total hits in microbatch 95 (paraffin/silicone)
Total hits in vapour diffusion 103
Conclusions Microbatch and vapour diffusion are
equally successful in screening for
crystallisation conditions, microbatch can be
used for volumes 200nl. Use both if you can
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Comparison of modified microbatch and vapour
diffusion
There are clearly differences between the 2
methods, do both if you can if you cant do
microbtach!
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Modified microbatch for screeningXray quality
crystals
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Microbatch and membrane protein crystallization

• There are very few reports of microbatch
  crystallization of membrane proteins, it may in
  fact be the system best adapted
• A recent articles by Loll et al. 20 and
  Dijkstra et al. 21 have shown that it is
  possible to use detergents in the microbatch
  system and
• A recent chapter in "Membrane Proteins" 22 by
  Naomi Chayen also gives some insights into the
  use of microbatch for membrane protein
  crystallization.
• Examples are given of membrane proteins that
  could be crystallised in the microbatch system.
• If screens containing detergents are performed
  under oil, the integrity of the drop is much
  better preserved than in vapour diffusion
  especially when plates are moved during
  observation, which is another positive aspect for
  screening membrane proteins for crystallization
  conditions.

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Recent Developments Fluidigm
The microchip method is a nanoscale free
interface diffusion that has a number of
similarities to microbatch, as no reservoir
solutions are used and a controlled, slow
evaporation of the drops allows high protein
concentrations to be sampled. Once conditions
have been identified using the microprocessor,
the microbatch method has been shown to be very
suitable for scaling up and optimising the
initial crystals.
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Mounting from microbatch no big deal!
Remove directly from drop using a cryoloop and
flash freeze in beam, oil will act as
cryoprotectant
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Mounting from microbatch no big deal!
Remove from drop with loop place in mother
liquor on glass slide (age of drop?)
Add reservoir solution to the drop if conditions
are known
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Why was microbatch not popular?

• Crystal stability 4-8 weeks in Als oil)
• Crystal handling and mounting
• Messy
• Skin formation
• Slower pipetting

• Solutions found

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So why is it worth a try?

• Easily automated (robot or multi-pipette).
• No sealing is required.
• Small volumes are possible with simple liquid
  handling devices (0.15ul -1ul).
• No reservoir solutions are needed (saves time and
  money).
• Suitable for temperature gradient experiments.
• Can provide a rapid screening method using
  silicone oil.
• Offers protection against protein oxidation and
  airborne
• contaminants.
• Reaches much higher concentrations of protein and
  precipitants.

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Microbatch references
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Glasgow, S. Fortier, and G. T. DeTitta J. Cryst.
Growth 232 (2001), pp. 591-595 Macromolecular
crystallization in a high throughput
laboratory-the search phase 2 R.C. Stevens.
Curr Opin Struct Biol 5 (2003), pp. 558-63 3
L.J. DeLucas, T.L. Bray, L. Nagy, D. McCombs, N.
Chernov, D. Hamrick, L. Cosenza, A. Belgovskiy,
B. Stoops, A. Chait. J Struct Biol. 142(1)
(2003), pp.188-206 4 B. Rupp 36(3) (2003), pp.
173-81 5. N.E. Chayen, P.D. Shaw Stewart ,
D.M Blow. Journal of Crystal Growth, (1992), pp.
176-180 6 N.E. Chayen., The role of oil in
macromolecular crystallization. Structure. 5(10)
(1997), pp. 1269-74 7 N.E. Chayen., Journal of
Crystal Growth (1999), pp. 434-441 8. A.
D'Arcy, C. Elmore, M. Stihle, and J. E.
Johnston.. J. Crystal Growth 168 (1996), pp.
175-180 9. N.E. Chayen, P.D. Shaw Stewart, P.
Baldock. New Developments of the Acta Cryst. D 50
(1994) pp. 456-458 10. N.E. Chayen, P.D. Shaw
Stewart, D.M. Blow. Microbatch. Journal of
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N.E. Chayen, P.D. Shaw Stewart, D.L. Maeder, and
D.M. Blow. An Journal of Applied Crystallography.
23 (1990), pp. 297-302 12. J. R. Luft, J.
Wolfley, R. Collins, M. Bianca, D. Weeks, I.
Jurisica, P. Rogers, J. Glasgow, S. Fortier, and
G. T. DeTitta. Proceedings of NATO ASI 30th
Course - Methods in Macromolecular
Crystallography, "Ettore Majorana" Centre for
Scientific Culture, Erice, Italy. (2000) 13. J.
R. Luft, J. Wolfley, I. Jurisica, J. Glasgow, S.
Fortier, and G. T. DeTitta. J. Cryst. Growth 232
(2001), pp. 591-595. 14. A. D'Arcy, A. Mac
Sweeney, M. Stihle, A. Haber Acta
Crystallographica Section D 59 (2003),pp 396-399.
15. B. Lorber and R. Giege J. Crystal Growth
168, (1996), pp. 204-215. 16. N. E. Chayen.
Comparative studies of protein crystallization by
vapor diffusion and microbatch. Acta Cryst. D54,
(1998), pp. 8-15. 17. P. Baldock, V. Mills, and
P. D. Shaw Stewart. J. Crystal Growth 168,
(1996), pp. 170-174. 18. N.Noordeen and S.
Cowan-Jacob Recent Advances In Macromolecular
Crystallization http//www.hamptonresearch.com/stu
ff/ppt_files/P6. (2001) 19. J.R Luft,. D.M
Rak,. G.T. DeTitta 196, Issues 2-4 15 (1999),
pp. 447-449 20. P. J. Loll, A. Tretiakova and
E. Soderblom Acta Cryst. D59, (2003), pp.
1114-1116 21. T. R. M. Barends and B. W.
Dijkstra, Acta Cryst. D59, (2003), pp. 2345-2347
22. N. E. Chayen, N.E. "Crystallization of
Membrane Proteins in Oils" Chapter 8 in "Methods
and Results in Crystallization of Membrane
Proteins", S.Iwata ed International University
Line, USA (2003) pp. 131-139 23. C. L Hansen,
E. Skordalakes, J. M Berger and Quake, S. R.
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