The overall goal of this procedure is to improve the number and diffraction quality of protein crystals obtained from a crystallization screening experiment. To do this, a crystalline or precipitated material is identified from an existing protein crystallization screening experiment established using a target protein of interest, the crystalline or precipitated material is harvested and then used to generate a crystal seed stock. Next, a series of crystallization screens is established containing both the target protein of interest and the crystal seeds.
Finally, the seeded crystallization screens are monitored for crystal formation and any new protein crystals are characterized. Ultimately, the application of this procedure can result in an increase of the number size and diffraction quality of protein crystals obtained from crystallization screening experiments. The main advantages of this technique over existing methods, for example, traditional micro seeding or macro seeding, are that it can be performed quickly, is highly scalable and sample significantly more crystallization space than other seeding methods.
Another advantage of this technique is that it does not require the use of high quality crystalline starting material for seed stock production. Generally, individuals new to this method will struggle because a significant degree of manual dexterity is required during the crystal harvesting and manipulation steps. Visual demonstration of if method's critical as random matrix micro seeding deviate significantly from other crystal seeding methods.
The subtleties of this method are key to a success, and a number of these are challenging to accurately communicate in text form. Only Begin this procedure with a seated crystallization tray using a binocular microscope or crystal imaging system. Inspect the tray each day for the first week.
Then once a week thereafter, make a note of crystal size. As soon as there is no evidence of further growth, select one or more appropriate wells from the tray from which to harvest crystalline material. For seed stock generation, any material can be used, including fine needles, spial lights, micro crystals, and irregular, poorly formed crystals.
Older material is more likely to be partially cross-linked and is less suitable for use in seeding experiments. On the day of harvest, use a buns inflamed to make a rounded probe from a glass pasture pipette. To do this, heat the pipette near the middle until it becomes soft.
Then quickly remove it from the flame and draw it out by pulling the ends apart. Aim to pull the glass down to a diameter of less than 0.25 millimeters at the point where it is about 0.25 millimeters. Break the glass and briefly plunge the broken end into the flame.
Repeat this process until a hemisphere of glass is formed on the end with a diameter of approximately 0.75 millimeters. Place a 1.5 milliliter micro centrifuge tube containing a seed bead on ice. Next, open the selected crystallization tray.
Well, for 24 well hanging drop trays. Use a pair of tweezers to remove the cover slip. Then invert the cover slip and place it on a stable clean surface.
For 96 well sitting drop trays. Use a scalpel to cut around the plastic ceiling Film on the top of the selected well and then remove the excised portion of the ceiling film using a pair of tweezers. Remove 50 microliters of the reservoir solution and transfer this liquid to the micro centrifuge tube containing the seed bead, ensuring that the tube remains on ice throughout The single most difficult aspect of this procedure is preparation of the seed stock.
It's important to perform this precisely as described and as quickly as possible. Take care to thoroughly crush the seed material with the probe and recover as much as you can While viewing the sample under a microscope. Use the glass probe prepared earlier to thoroughly crush the crystals on the cover.
Slip drops. Small crystals may take several minutes to crush thoroughly. Crystals that are easy to crush are not cross-linked and are not salt crystals.
While salt crystals produce a distinctive click that can be heard and felt when they're crushed. Next, remove five microliters of the reservoir solution from the seed bead tube and transfer to the equivalent sub well for a sitting drop experiment or cover slip for a hanging drop experiment containing the crystalline material to be harvested. Pipette this liquid up and down five to six times to resuspend as much of the sub well or cover slip contents as possible.
Return the suspension to the seed bead tube and repeat this step twice more, ensuring that as much crystalline material is harvested as possible. Vortex, the seed bead for two minutes, stopping every 30 seconds to cool the tube on ice. In 1.5, milliliter tubes containing reservoir solution.
Make a dilution series to archive diluting the seed stock in reservoir solution by a factor of four to 10 at each stage. If the seed, stock and dilutions will not be used immediately, place 10 to 20 microliter aliquots in a negative 20 degrees Celsius or negative 80 degrees Celsius freezer for storage. Once frozen, the seed stocks may be kept indefinitely until needed.
RMMS crystallization screening can be performed using either 24 well trays employing the hanging drop vapor diffusion method or 96 well trays using the sitting drop vapor diffusion method. To perform our MMS screening in 24 well hanging drop trays use a pipette to manually transfer 300 microliters of each condition from a 96 condition crystallization screen in 10 milliliters. Single tube format to each well of 4 24.
Well pre-greased crystallization trays from each 300 microliter reservoir transfer one microliter of each crystallization condition to the surface of a corresponding plastic cover slip from which both front and rear protective backing strips have been removed to the one microliter. On the cover slip, add one microliter of protein solution, then 0.5 microliters of crystal seed stock. In the first round of RMMS, it is important not to use the diluted seed stock solution.
Since the greater the concentration of seeds, the more crystallization hits will be obtained. Invert each cover slip such that the drop of liquid is downward facing and position above the appropriate. Well press downwards on the cover slip, compressing the ceiling grease, and forming a secure seal.
Once all 96 drops are established, transfer the tray to either an incubator at four to 18 degrees Celsius or constant temperature room for storage. To perform our MMS screening in 96, well sitting drop trays. Use a crystallization robot or an eight channel pipette to transfer 20 to 50 microliters of each condition from a 96 condition crystallization screen in deep well block format into each corresponding well of the crystallization tray.
Next, transfer, 1.0 microliters of protein solution, 1.0 microliters of crystallization condition and 0.5 microliters of seed stock to the drops. Seal the tray using a transparent ceiling sheet and transfer it to an incubator at four to 18 degrees Celsius or constant temperature room for storage experiments should be inspected once every 24 hours for five days following establishment, and then subsequently once every seven days for up to four weeks using a binocular microscope or a crystal imaging system. Inspect each sub well or cover slip in sequence and record any evidence of crystal formation.
Compare the results from our MMS screening experiments with those from non-ED screens and record any differences. Observed multiple cycles of RMMS may be required. Before optimal diffraction quality crystals are produced for additional cycles.
Use the archived diluted seed stocks as needed to control the number of crystals per well. To demonstrate the effectiveness of RMMS. Screening methods were applied to the crystalization of henna, white lysozyme, and bovine liver catalyst for each protein 3 96 condition.
Crystallization screens were used J-C-S-G-P-A-C-T, and Morpheus. Then after five days, a single condition from each tray that was found to support the growth of crystalline material was selected and used for seed stock generation. After five days, the crystallization trays were inspected and the number of drops with crystals was recorded.
This figure summarizes the results from this experiment and provides quantitative analysis for hen egg white lysozyme. A four to tenfold increase in crystallization success rate as compared to non-ED trays was observed when RMMS was applied to this protein. Notably for bovine liver catalase only a single condition in the Morpheus screen was found to yield crystals as compared to 55 conditions identified following the use of RMMS.
Further, there was a three and twofold increase in the success rate of bovine liver cans crystallization using JCSG and PACT screens respectively in all cases, and irrespective of the crystallization screen used, our MMS screening yielded a significantly greater total number of crystals than non RMMS screening. We conclude that our MMS is a simple yet powerful approach, which can significantly increase the success rate of protein crystallization screening experiments. After watching this video, you should have a good understanding of how to perform a random micros seeded matrix screening experiment from the identification and harvesting of crystalline material through the preparation of crystal seed stock, and finally, the establishment of seeded crystallization Once mastered.
This technique can be done in less than one hour. For random matrix micro seeding screening experiments performed using a liquid handling robot or in around three hours for experiments performed by hand. While attempting this procedure, it's important to ensure that all seeded crystallization screens are well sealed prior to storage.
