Tips, Tricks and Troubleshooting

Seeding progession from a single crystal drop without seeding to an optimised crystallisation condition for XChem using seeding.

Seeding

The use of seeding in protein crystallisation can be crucial for obtaining crystals. Using seeding can become necessary for producing protein crystals in conditions that previously did not require seeding. This is often due to issues in the quality of protein samples and crystal conditions after transportation or differences caused by being used in a different laboratory.  However, the process is still applicable in cases where crystals can be grown without seeding as a method of increasing the quantity of usable crystals from a single plate. An example of the use of seeding can be seen above.  

Once made, a seed stock can undergo multiple thaw freeze cycles without losing effectivity. After thawing a seed stock for use in plate setup, the stock should be spun down for ~10 seconds to ensure the distribution of micro crystals is consistent throughout the stock solution, to maintain the consistency of seed concentration in drops. Stocks can be easily contained in a single falcon tube to ensure all seed stocks are together and easily locatable and transportable.  
 
When in use, the volume of seed stock used in the protein drop is taken off the volume of crystallisation condition used e.g., if a drop uses 100 nl of crystallisation solution, this may change to 80 nl of crystallisation solution and 20 nl of seed solution. The changing the volume of seed solution used in the drop will change the number of crystals grown. Along with the production of a more specific seed dilution, if necessary, these changes allow for further optimization of the crystal growth after the introduction of seeding. 

Protocol: 

1. Produce crystals to use for seeding. Micro crystalline protein material can be used to create a seed stock as well. 

2. Set up the serial dilution for step 10 using the reservoir solution used to obtain the starting crystalline material on ice. Make up an appropriate volume; most seeding uses 20-50 nL of seed stock, meaning a SwissCI 3 lens 96 well plate could require ~14.5 µL of seed stock per plate.

3. Add seed beads to the undiluted tube. The number of seed beads required is based on the size used, so add a number of beads based on the instructions of the product. 

4. Under a microscope, open the well containing the crystal material being used and crush the crystals.
   From this step onwards proceed rapidly as the seed stocks are metastable and need to be frozen as soon as possible. Having the serial dilution already set up and chilled is important in ensuring the following steps can be carried out without the need to flash freeze the seed stocks after creation. 

5. Pipette 2 µl from the reservoir well into the drop well. 

6. Mix the solution by aspiration a minimun of three times. 

7. Transfer to the first tube set up for the serial dilution that will remain undiluted and is marked "Undiluted seed stock". 

8. Repeat steps 5-7 until there is no more crystal in the drop well. To ensure all available crystal material is recovered, a minimum of around 30 µl of reservoir solution should be used in this process. 

9. Vortex the undiluted tube containing the seed beads – 30 seconds on vortex then 30 seconds on ice, repeating three times. Take 2 µl of the seed stock and check under microscope whether the crystals have been crushed.

10. Carry out serial dilution using the undiluted seed stock and freeze stocks at -80 ^oc . 

Using seeding to find diverse crystallisation conditions and crystal forms: 

• Achieved using Microseed Matrix Seeding (MMS), which is the addition of seed solution made from one crystallisation condition, which is then added into the contents of a crystal screen.  

• Crystals obtained can; form into different space groups, have better resolution, help remove problematic crystallisation conditions e.g., isopropanol. 

Establishing size variations of crystals: 

• Achieved by altering the volume of seed solution and dilution of seed solution used in seeding; this will be unique to each crystal system. 

• Want a variety of sizes and shapes in case smaller crystals cannot withstand sample conditions for shooting as well as potentially dissolving due to cryoprotectant. 

• The results can inform on if the crystal system used in screening needs optimising compared to the results of the solvent test. 

Establishing a final protocol for high-reliability growth: 

• Having reproducible crystals to make sure the current and future screening of fragments and follow up compounds is consistent. 

• When necessary, seeding can take place in iterations to further increase the quality of crystals obtained by making new seed solutions with results obtained from seeding. 

• The use of seeding makes crystal systems highly reliable, especially when following the seeding protocol to ensure consistency when doing iterations of seeding. 

Transferring crystallisation conditions to another laboratory: 

• If you are sending a crystal system that uses a seed stock, the solution will need testing. Even if seed solution is not needed in the home lab, it is still important to create and send a seed stock as many crystal systems work perfectly in the home lab and then have troubles being reproduced at XChem. 

• Testing the seed stock before sending will ensure that crystals can be obtained easily. It will help increase the consistency of results, to make sure your data doesn’t miss potential binding due to poor crystals. 

• The home lab should simulate shipping by trying with fresh seed stock and frozen, so use fresh, then freeze, then thaw and try again. If using a diluted seed stock, also send some of the original undiluted seed stock to account for differences in crystallisation in different laboratories. 

Further reading: 

https://www.douglas.co.uk/MMS_proc.htm 

https://www.douglas.co.uk/f_ftp1/rMMS_Procedure.pdf