Advantages of t method in sccrystallizatio

Aengus Mac Sweeney1, Allan1Morphochem AG, Basel, Switzerland

2Hampton Research Corporation,

he microbatchreening forn conditionsD’Arcy1 & Bob Cudney2

Laguna Niguel, USA

100nl

200nl

400nl

Drops of dyed protein and 25% PEG 3350 on Terazaki plate. Pipetted onto plastic before addition of oil

100nl drop of aldolase with crystals at low (A) and high (B) magnification

A B

• Using a Douglas Instruments robot it is possible to reproducibly produce drops of 100nl (50nl protein + 50nl reservoir).

• In order to pipette accurately the drop must be allowed to adhere to the plastic of the plate in the absence of oil, then be covered immediately with oil to minimise evaporation.

• The figures below show drops pipetted directly onto a plastic surface (Terazaki plate) using a Douglas Instruments robot, then covered with oil.

Low Drop Volume - Save Protein - Larger Screens

•Low temperature and temperature gradient experiments can be used as an additional variable in crystallisation screening or as an optimisation tool.

•Vapour diffusion experiments are not suitable for temperature gradients because condensation tends to form on the inside of the coverslip.

Temperature Variation of Microbatch Screens

Hampton research M6 incubator

Microbatch allows easy temperature variation

ReservoirReservoir

~20 umoles O2

~1 nmole protein Vapour Diffusion•Protein drop exposed to air•Reducing agent may be required

Microbatch•No direct exposure to air•Protein more likely to crystallisebefore oxidation occurs

Microbatch protects the protein against oxidation

• Screens of 18 crystallisation conditions were set up

• Using the Hampton Research incubator, the screens were stored under various temperature conditions

• The screens were scored after 24 hours and 3 days, no extra crystals were observed between 24 hours and 3 days

• A gradient of 30° to 4°C over 16 hours was the most suitable for crystallization

An example with Aldolase

Crystals of aldolase grown at different temperatures (identical solutions)

• The results shown above were obtained using aldolase with18 conditions from the standard crystallisation screen.

• When a similar screen of approximately double the precipitant concentration was used, more crystals were obtained at all temperatures but the 30° to 4°C gradient remained the best.

Protection against oxidation

• Oxidation of proteins, can cause nonspecific aggregation and prevent crystallisation.

• In a vapour diffusion experiment the drop is exposed to air within the reservoir. The crystallisation drops in a microbatch setup are shielded by a layer of oil through which oxygen diffuses slowly, increasing the probability that the protein will crystallise before oxidation occurs.

• In one instance we have crystallised a protein using microbatch screening and have not been able to obtain crystals using vapour diffusion

•We are now investigating oxidation of the protein as a possible cause of its failure to crystallise using vapour diffusion

Drops of dilute permanganate (<1uM) before setup (A), after 16 hrs. microbatch (B)and after 16hrs. hanging drop (C) (Microbatch drop transferred to glass slide for picture)

• To compare exposure to air, 4ul drops of potassium permanganate were made in hanging drop and microbatch format.

• Almost no colour change could be detected in the microbatch drop. The vapour diffusion drop changed to a yellow/brown colour overnight, indicating exposure to air.

•The experiment was repeated with ferrous sulphate and the same effect was observed

Initial drop

Microbatchdrop

Hanging drop

Addition of precipitant Plate after centrifugation

Manual microbatch setup1. Oil pipetted into wells of 96 well plates (Figs. 4A, 4B)2. Reagents pipetted using 8 channel pipette (from premixed stock)3. Samples pipetted using single channel pipette4. Plates centrifuged (5 minutes, 2500 rpm) to coalesce drops

Five screens in 45 minutes:>40 screens (4,000 drops) per 8 hour day

Cost of hardware: $7100(Centrifuge $2500, Oil machine $1500, Pipettes $600)

High Throughput, Low Cost Crystallization

Examples of two proteins that require microbatch crystallizationMicrobatch Vapour Diffusion

Identical solutions used in both cases

Conclusion

Microbatch screening• uses less protein• is more suitable for temperature gradient experiments• can be made high throughput at low cost• offers some protection against protein oxidation