Tutorial 5

Seeding is the #1 optimization tool to use. If you have any kind of crystal, (or even gel, or precipitate), it means you are in the supersaturated zone of the phase diagram. So, use this solid phase, however unpromising it might look, as a seed. . I cannot emphasize this enough. Sure, you can fine-tune the pH, the precipitatant and protein concentrations, etc., etc. to improve your crystals, but it's far easier and more efficient to seed from what you have found in the supersaturated zone.

There are different kinds of seeding: macroseeding, microseeding, streak seeding, epitaxial seeding, cross-seeding, matrix microseeding, heterogeneous and homogeneous.

Nowadays I exclusively use matrix microseeding with a robot to do my seeding. It's the fastest and easiest way to optimize. Read more here. This paper gives full details on how to make the seed stock, but if you want to cut to the chase,  download the step-by-step protocol here.

Need more tips on seeding? Click here.

Or watch this lecture video  (13 min) explaining how I use matrix microseeding.

Below are some visual examples of just how powerful seeding can be.



Matrix Microseeding


These crystals appeared in a condition from the Morpheus screen.They are about 20 microns, inside a phase separation. I used the entire drop to make a seed stock, then reseeded into a new plate, also containing the Morpheus screen.


Matrix Microseeding

Within one day, these crystals appeared (seen here with UV) in the same condition that produced the crystals shown above. These diffracted to 1.6 Å and we solved the structure of Pseudomonas aeruginosa IspC with a single round of optimization.

To learn more, watch this video.

Microseeding step 1. CBH II Example

These crystals of cellobiohydrolase II grew from a single nucleation site. Notice that you also have a phase separation in the drop. To learn what that is go to tutorial 3.

We wanted to improve the quality as well as grow them as single crystals. Therefore these crystals were crushed and suspended in a slurry of mother liquor to make a “seed soup” or “seed stock”.

Microseeding step 2. CBH II Example

A new drop was innoculated with microseeds from the slurry. Centrifuge the slurry for 30 seconds at 1-2 g. Take an acupuncture needle, and dip it into the slurry. Now touch the new drop with the needle. The experiment worked as you can see. The crystals now grow singly.

Microseeding step 3. CBH II Example

The cycle of microseeding was repeated again, using the new, improved crystals obtained in step 2. This crystal is the result. (It is over 2 mm long).

Microseeding step 1. Acyl coA Binding Protein (ABP) Example

Here is another example of using seeding to improve crystal quality. These crystals grew in an enormous number of conditions, but as masses of needles. These were used as microseeds.

Microseeding step 2. ABP Example

After microseeding into a new drop, the crystals grew singly. Alas, they are still only thin needles.

Microseeding step 3. ABP Example

Microseeding doesn’t solve everything. Seeding did not produce anything but needles. Cation choice was decisive in changing the crystal habit. Over 20 cations were tested as additives. All gave needles except this one, nickel sulfate.

Microseeding step 4. ABP Example

When a ligand was added, these crystals grew. These were the ones that were with the best diffraction but took 4 months to grow. You can speed up the process considerably by using seeds of the apo-protein to seed the drops containing the holo-protein. To grow these crystals we had to use three optimizations: seeding, the cation Ni, and the ligand.


Streakseeding is a manual method although Douglas Instruments does have a script for doing it on their Oryx robot.

Enrico Stura first published about it in 1992.

Take a cat whisker. Stroke the parent crystal to pick up invisible seeds. Now, draw a streak line across a new drop. Crystals will grow along the streakline. Sometimes you will also get new self nucleated crystals in parts of the drops farther away from the streakline.