Optimisation of Initial Hits
Initial hits from broad screens can range from micro crystals, needles, plates, clusers/sea urchins and even single crystals ready for data collection. This tutorial from Teres Bergfors gives examples with pictures of each of these cases. If you aren't lucky enough to get a single crystal first time then it's time to start optimising. To do this it helps to have an understanding of what's going on in your drop, and the best way to visualise this is using The Phase Diagram.
A vapor diffusion crystallisation experiment works by gradually increasing the concentration of protein and precipitant as water diffuses out of your drop into the reservoir solution (mother liquor). As your protein and precipitant approach super saturation the drop will (hopefully) enter the nucleation zone, where your protein will fall out of solution and form small crystals. The protein concentration in solution will then decrease and the drop will transition into the metastable zone where new crystals stop forming and crystals being to grow. By observing your inital hits in relation to the phase diagram we can get a better idea of how to get to single crystals by altering initial protein/precipitant concentrations.
The easiest way to do this is to set up a custom 96 well plate with a precipitant and pH gradient based on the conditions that gave your initial hit. The facility has a Dragonfly Liquid handling robot to help facilitate setting up hit optimisation in MRC plates with the Mosquito robot.
Seeding
If your optimisation plate gives you more of the same (clusters, needles, sea urchins, etc) then don't dispair. These suboptimal crystals can be used as seeds for another optimisation plate. By adding seeds to our crystallisation drop we can bypass the nucleation step and go immidiately into crystal growth.
You can make seeds by following this protocol:
- Harvesting several drops of crystals from the tray with a pipette
- Add glass beads to the crystals and votex
- Make a serial dilution of your concetrated seed stock (10, 100, 1000, 10000, etc)
- Redo the optimisation tray using one of the seeding programs on the Mosquito with a seed stock gradient
