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Protein Crystallization Robot for sitting drop and
microbatch screening with (simple) optimization
|Oryx Nano, 4 and 8||Oryx4 and 8|
|Sitting drop screening||Hanging drop|
|Drop volume range||Additive screening|
|"rMMS" and regular microseeding||Microbatch-under-oil|
|Minimal protein wasted||Oryx4 can be upgraded to Oryx8|
|Simple 2D gradients||Lipidic Cubic Phase (LCP) option|
|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.
Features of the Oryx4 and Oryx8 systems only:
Oryx4 and Oryx8 can set up experiments with 22 mm and 18 mm cover slides as well as 15-well Qiagen EasyXtal plates, with up to 5 drops on each cover slide. Volumes can range from 100+100 nl up to 8+8 µl (assuming 24 drops). You can also add one additive to each drop with Oryx4, and up to five additives with Oryx8. This is very useful for leads that are picked up in random microseeding experiments, where it may be necessary to add e.g. diluted seed stock to get crystals.
For example, in the experiment shown (below below), one drop has seed-stock, protein, reservoir solution; the second drop has no seed but a little extra protein, while the third drop has no seed but a little extra reservoir solution.
You have to transfer the cover slides onto the reservoir tray by hand. After a few drops the experiment can be paused to allow the transfer. Oryx8 can set up the reservoirs for optimization experiments, including 2D grids and multivariate experiments (see below). The hanging drop capability is not available for the OryxNano.
Additive experiments are a well-known and effective approach to optimization. Simply by adding a set of potential ligands or another screen to a hit condition, crystals can often be optimized. Since they have larger Plate Loaders, the Oryx4 and Oryx8 systems can accommodate an extra 96-well plate containing additives to the right of the target plate. The tip picks up e.g. 100 nl samples from the additive plate and dispenses them along with protein and reservoir solution to the drops. For initial screening relatively large volumes of additive are often used, for example 300 nl (protein) + 100 nl (reservoir) + 200 nl (additive). For example, these volumes could be used for an initial screen with the Silver Bullet screen by Hampton Research. For final optimization, smaller volumes of additive are often used, for example 300 nl (protein) + 200 nl (reservoir) + 100 nl (additive).
Microbatch is a very simple approach to crystallization. Small samples of protein (100 nl to 5.0 µl) are mixed with stock solutions in small drops, and covered with oil to prevent evaporation. For screening experiments it helps to use a 50:50 mixture of paraffin oil and silicone oil. The silicone allows slow evaporation over about a month, which gives a scanning effect across the phase diagram of the protein. For optimization, pure paraffin oil can be used, which reduces evaporation to a minimum. Studies have shown that microbatch finds as many or slightly more hits that vapor diffusion, but the main advantage is that (for reasons that may not be well-understood) certain proteins crystallize much more effectively in microbatch than other methods. Microbatch can help to protect sensitive proteins such as membrane proteins and anaerobically-produced proteins because it reduces the oxidation and gives thinner skins on the surfaces of drops.
A full set of experiment scripts for microbatch are available, including screening, additive screening, 2D gradients and MMS microseeding. Oil is automatically dispensed onto the aqueous drop covering it immediately.
Using the Combined MB-VD experiment it is possible to dispense two screening methods at the same time. It is also possible to dispense different drop ratios or add seed-stock or additive to any experiment or drop.
The Oryx4 system can easily be upgraded to Oryx8 simply by adding a second Chassis and changing the colors of the connectors. However, the OryxNano cannot be upgraded easily because it has a smaller Plate Loader. Contact the company for more details.
Lipidic Cubic Phase (LCP) dispensing is an option for Oryx4 and Oryx8 (not available for Oryx Nano). The option is available for new systems and can also be installed to recent compatible systems. An arm mounted positive displacement syringe drive can dispense LCP volumes as small as 10 nl and up to 1 µl or larger. The system can dispense to LCP sandwich plates such as Laminex, Marienfeld and IMISX 96 well plates. The system can also dispense LCP to all other compatible plates, including sitting drop plates.
The shape of the volume (dispensing technique) can be selected in the software. This allows the choice of taller conical volumes or wider pancake shaped volumes (see image below). After the LCP volume is dispensed the precipitant(s) and additives are immediately dispensed covering the LCP volume and preventing evaporation or phase change of the LCP.
Oryx LCP machines are not able to dispense under oil or vapour diffusion optimization experiments. This is because the LCP dispensing arm is in place of the large volume dispensing arm. However it is possible to reconfigure the machine between large volume dispensing and LCP dispensing modes in approximately 15 minutes.
Oryx4 and 8 LCP can dispense LCP to 22 mm or 18 mm hanging drop coverslips. This offers much better harvesting than a sandwich plate and better optics than sitting drop.
The Douglas Instruments manual LCP mixer is supplied with all Oryx LCP machines. It allows simple preparation of LCP mixtures and because it is manually operated the user maintains kinaesthetic awareness of the sample during preparation. The mixer is also available from Jena Bioscience.
Oryx4 LCP can dispense normal LCP screening and additive screening experiments. **We will soon add dedicated experiment scripts for cross matrix optimization and simple 2d grid optimization LCP experiments.
** coming soon.
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