This is the detailed information for workshop #5, Crystallization, by Bergfors, Erice 2010
The purpose of this experiment is: 1. to learn how to generate new seeds by the easy, fast, and simple method of streak seeding; and 2. to observe the effects of decreasing protein concentration on the nucleation rate.
This workshop is suitable for complete novices, even if they have never set up crystallization plates. It is also suitable for those who have at least the basic skills of setting up vapor-diffusion drops and would like to learn more about seeding as an optimization method. The exercise will provide the participants hands-on training in the fast and easy method of manual streak seeding. Seeding can also be implemented with a robot, for example with the Oryx (Douglas Instruments) and others. The workshop leaders will discuss how to recognize phase separation, nucleation, and other drop phenomena seen in the laboratory exercise. We will also have on hand different loop types for cryocooling and various microtools for manipulating crystals.
Useful background reading before the workshop:
- Bergfors, T. "Seeds to Crystals" J. Structural Biology, 2003, vol. 142, 66-76.
- D'Arcy, A., Villard,F., Marsh, M. "An automated microseed matrix screening method for protein crystallization", 2007, Acta Crystallographica, D63, 550-554.
- Bergfors, T. "Succeeding with Seeding: Some Practical Advice", 2007, Evolving Methods for Macromolecular Crystallography, Eds. Read, RJ and Sussman, RL, Springer Press, 1-10.
The experiment takes 30 min to 1 hr. Each person may set up on his/her own individual experiment. We will work in groups of 4 people per microscope.
These supplies will be provided by the workshop organizers. 1. Gilson pipette that can pipette a volume of 10 microliters 2. Gilson pipette (P2 model) to pipette 0.5 - 1 microliter. 3. Plenty of tips for the above pipettes 4. Seeding Tool Hampton HR8-133 5. stereo microscopes 6. Petri dishes (the big kind that hold 20 ml) 7. marking pen for writing on the Petri dishes 8. test tube racks capable of holding Eppendorf tubes 9. bag of eppendorf tubes, 1.8 ml size
Protein solutions required: These supplies will be provided by the workshop organizers. 1. 1000 microliters of 100 mg/ml lysozyme in water as 4 x 250 ul aliquots 2. 1000 microliters of 80 mg/ml lysozyme as 4 x 250 ul aliquots 3. 1000 microliters of 60 mg/ml lysozyme as 4 x 250 aliquots 4. 1000 microliters of 40 mg/ml lysozyme as 4 x 250 aliquots 5. 1000 microliters of 20 mg/ml lysozyme as 4 x 250 aliquots
Other solutions required: These supplies will be provided by the workshop organizers. 7. 10 ml of 30% PEG 6000, 1M NaCl, 50 mM Na acetate, pH 4.7. 8. 2 x 250 ml of 6% NaCl in 50-100 mM acetate buffer, pH 4.7. 9. 2 x 250 ml of 12% NaCl in 50-100 mM acetate buffer, pH 4.7.
Comments: • Which brand of lysozyme? Merck # 1.05281 is the best, but Sigma, Serva, etc. should all be okay. • Dissolve the lysozyme in water well in advance of the workshop (even several weeks ahead is fine. The older the lysozyme solution, the better for this experiment). Do not filter it. Put it in eppendorf tubes. It can be kept at 4 degrees C., but it is not absolutely required.
Here are the step by step instructions for doing the workshop Method:
1. Make the parent crystals which will serve as the seeds. On a petri dish lid, cover slip, etc, mix 10 ul of lysozyme (100 mg/ml) with an equal volume of the precipitant (= 30% PEG 6000 in 1M NaCl, 50 mM Na acetate, pH 4.7).
2. Crystals should nucleate in 5-15 minutes. The fresher the lysozyme the longer time it will take to nucleate.
3. Observe under the microscope. It can take some practice to recognize the nucleation in its initial stages. Note: If the protein precipitates immediately, the protein concentration is too high. Redo with a slightly lower protein concentration; try 80 mg/ml.
4. Prepare the four droplets that will be seeded. While the crystals in step 3 are nucleating, you can begin preparing the dilution series where you vary the concentration of the lysozyme. Set up 10 ul drops of lysozyme at 80, 60, 40, and 20 mg/ml. This is easiest to do directly on the cover slip or the lid from the 24-well plates:
5. Add 10 ul of the precipitant to each of these 4 new drops and stir.
6. Now you can transfer by streak seeding the nuclei generated in step 3. Dip the seeding wand once into the drop of parent crystals to pick up the seeds, then streak the wand across the four new drops. You do not need to re-dip the wand into the parent drop before each new streak; there are plenty of seeds on the wand for all four droplets.
What to look for or think about: 7. See if you can get the crystals to grow along a streak line. Because of the high protein concentrations you will also get spontaneous nucleation but many crystals will form preferentially along the streak line. You should get fewer, but larger crystals as you decrease the protein concentration.
8. The experiment lasts only 30 minutes. As a consequence, the crystals produced will not be very large, but the experiment will illustrate the procedure for seeding and this can be applied in real cases back home.
Here are the "bonus projects" you may work on if you finish the workshop early.
- 1. Growing red lysozyme crystals in microfluidic chips. We will provide microfluidic chips (www.microlytic.com) and demonstrate how to use them. The lysozyme crystals usually grow in about one hour.