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Protein Crystallization Robot for sitting drop screening
with (simple) optimization
|Oryx Nano, 4 and 8|
|Sitting drop screening|
|Drop volume range|
|"rMMS" and regular microseeding|
|Minimal protein wasted|
|Simple 2D gradients|
|Cross-Matrix (additive) optimization|
|Additive scatter optimization|
All Oryx systems use multi-bore dispensing tips (microtips), which have several independent channels to dispense small volumes. At the end of e.g. a 3-bore tip there are three holes. Each channel dispenses a different solution. The solutions do not mix in the tip - they mix in the drop after they are dispensed. This means that there is no dead-volume. Oryx8 has two chassis and can use 3, 4 and 7-bore microtips. The microtip is on the left-hand arm.
• The microtip always touches the plate when liquids are dispensed. This gives very reliable dispensing, especially when suspensions of e.g. seed crystals are used, which makes the system ideal for microseeding experiments.
• Stock solutions do not come into contact with the motorized syringes, which are filled with degassed pure water - this avoids the need for flushing out the syringes when the stock solutions are changed.
• The manual syringes on the front panel are used to refill the motorized syringes, to remove air bubbles, and to load stock solutions for optimization experiments.
• Large volumes, including oil for microbatch experiments, are dispensed by the right-hand arm using the large (2 ml) motorized syringe. A 1 ml disposable tip is used for this.
Screening for sitting drop is carried out using plates that are prefilled with reservoir solutions. (Reservoirs can be filled manually with a 12-channel pipette, or large-volume dispenser such as the Liquidator 96 by Rainin). During each dispensing cycle, the tip is first cleaned in the reservoir by moving it horizontally through the solution. The tip then picks up e.g. 100 nl of solution from a clean part of the reservoir and transfers it to the drop, dispensing protein simultaneously (together with seed stock for microseeding experiments). The level of contamination has been tested and is very low.
A very simple user-interface is used to design experiments, as shown below. The correct plate can be selected from a database of all well-known crystallization plates (if your favorite isn't there, let us know!), the volumes of each ingredient of a drop are entered in a simple form. Individual wells can be selected or skipped by clicking on the plate with a mouse. (The form shown below allows seed stocks and additives to be added to drops, as explained below.)
Exactly the same mechanism is used to dispense drops up to 8+8 µl for crystal harvesting and soaking experiments, and down to 100+100 nl for screening experiments. Smaller drops can be used but will not give reliable results with some proteins. Evaporation is reduced by covering the plates with a sliding shield (see below). For soaking experiments, microseeding is highly recommended, see below. For information about dispensing reservoirs for optimization experiments, see below.
Sliding evaporation shield
For sitting drop experiments a sliding evaporation shield is required (see right).
The shield acts as a cover for the plate and allows vapor pressure to build up.
This prevents evaporation from the drop in the same way as applying a plate seal. No extra humidification or temperature controls are required.
Since all Oryx systems use contact dispensing (the tip always touches the plate) they give very reliable dispensing even when suspensions of solid particles are used. This makes them ideal for microseeding experiments. The technique of adding crystal seed-stock to random screens (rMMS) is a significant breakthrough in protein crystallization that is very effective. For example, one industrial group used the method to solve 38 out of 70 structures generated in a four year period, finding particular success with antibody complexes. rMMS not only produces more hits, it also typically generates better-diffracting crystals – because crystals are more likely to grow in the metastable zone of the protein’s phase diagram (see below).
Note also that in cases where only one or a few crystals are obtained in screening experiments, the seed stock that can be made is very valuable – often more valuable than the protein sample. It is therefore a great advantage to be able to use the smallest possible sample of seed stock. Using any robot from the Oryx range, seeding can be performed in a whole 96-well plate using only 1.5 µl of seed stock. This is particularly helpful for membrane protein crystallization projects because membrane protein crystals are often unstable and it is helpful to make seed stocks without diluting the original mother liquor.
Contact dispensing has another advantage: almost no protein remains in the tip at the end of the experiment. Moreover, since only one (multi-channel) tip is used, all of the protein for an experiment can be placed in a single PCR tube, which also reduces waste. When they have enough protein, most users set up 300 + 300 nl drops. For a 96-well plate this requires only 29.4 µl of protein, i.e. only 0.6 µl is wasted. If your pipette is accurate, there is no need to put more than the specified amount into the tube!
Similarly if 10 nl of seed stock is added to each drop, only about 1.5 µl of seed stock is required for a whole 96-well crystallization plate. (It is helpful to dispense around 5 µl of screen solution on top of the seed stock.)
All Oryx systems can carry out simple optimization experiments using three different approaches.
The standard screening software allows users to define simple 2D grids for sitting drop, *hanging drop and *microbatch-under-oil experiments (*Oryx4 and 8 only). These experiments use three or four ingredients, the user specifies the minimum and maximum concentrations to be dispensed to the corners of a rectangular grid. The software interpolates linearly between those conditions. (The user interface does not show the concentrations in intermediate wells, but the volumes dispensed to each well are shown in a report file.)
For vapor diffusion experiments, the robot dispenses ingredients to the reservoir well first, then loads protein and other precious solutions such as seed stock before dispensing all ingredients to the drop well simultaneously. Note: Oryx Nano robots do not have a large volume dispensing channel and therefore can only dispense smaller volumes of ingredient solutions to the reservoir well. Oryx Nano robots typically dispense a total volume of up to 20 µl to each reservoir well. Oryx4 and 8 robots typically dispense a total volume of 20 - 500 µl to each reservoir well.
(A more sophisticated approach to optimization that includes gradient experiments, multivariate optimization and reservoir filling with a greater number of ingredients is available with the Oryx8 – see below.)
Videos of 2D grid experiments:
The systems' powerful “combinatorial optimization” approach allows a different additive or seed-stock to be added to each row. Each additive is picked up from the corresponding PCR tube on the right of the table (A1, A2 etc. on the diagram below). By arranging e.g. precipitants in columns (P1, P2 etc.), different combinations of precipitants and additives can be tested very quickly. This is useful for reshuffling the ingredients of several hits, so that ingredients that are not helpful can be eliminated quickly, and trends can be identified. For example, certain ingredients may encourage the formation of crystals with certain morphologies.
The combinatorial approach can also be used to systematically identify the appropriate dilution of a seed stock in a single experiment. We recommend using a highly concentrated seed stock for routine rMMS screening, however this can result in showers of small crystals. It is often possible to optimize these conditions by diluting the seed stock to get around 5 crystals per drop (experiment with thermolysin shown above). For example, different concentrations of seed stock could be placed in the PCR loading tubes shown 1 - 1E-6 dilution above. Four different conditions could be placed into the four columns labeled P1 to P4 above.
This is a very effective way to get a really reliable supply of crystals for data collection and soaking experiments.
Video about Cross-Matrix Optimization
Scatter up to 5 additives (e.g. seed stocks) evenly distributed across a vapor diffusion plate. The vapor diffusion plate would typically be pre-prepared with a 2D gradient of precipitant against salt or precipitant against pH. The robot will then distribute up to 5 additives in a pattern across the 2D gradient. This tests the additives across a range of concentrations.
This experiment would normally be used for testing up to 5 dilutions of seed stock. It could also be used to test other additives or protein concentrations.
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