You can try micro-seeding

I am working on small molecules crystallization and I used to get big crystals when I take saturated solution for crystallization. But I dont know for protein crystallization whether the crystallization technique is same or not?

There are a number of questions that need to be answered. (1) How long does it take to obtain crystals, (2) do you have the X-ray structure? (3) Is the full length protein crystallizing or is it a fragment? (4) Do you prepare buffers everytime or do you work with stock solutions etc etc

Depending on the answers, you can design an experiment to obtain large crystals reproducibly.

If too hard to get single crystals, you may try high resolution powder neutron diffraction

Yes, seeding can help. I did grow crystals large enough suitable for neutron diffraction but never collected the data.

Dear Mahfuza,

I understand that you would like to obtain, on a regular basis, large crystals with approximate volume of around 1mm^3, amenable for neutron diffraction. Alexander is absolutely correct that you need to check all the small details.

However, there are some general advice you can follow to improve your chances.

There are three dominant reasons why crystals stop growing:

1) exhaust protein material in solution (change of relative saturation of protein versus the precipitant),

2) accumulate sufficient amount of impurities on the surface that they reach equilibrium (impurities can be just simple vacancies or dislocations),

3) your crystals are topologicaly imperfect (they stop growing because of a misfit at larger distances).

Protein crystals are fundamentally different from crystals of small molecules. There are three dominant differences:

1) protein crystallization is mostly driven by entropic gain (solvent driven), while small molecule crystals are driven by enthalpic gain (packing that usually excludes solvent), so even small changes in salt concentration or the temperature would matter a lot,

2) they have, on average, more than 50% solvent (water) content,

3) the intermolecular forces in small molecule crystals are comparable to intramolecular forces, while in protein crystallography intramolecular forces are approximately 10 times stronger then intermolecular forces (solvent contribution).

There are simple consequences of both sets of facts. Small molecule crystals are on average more topologically ideal which results in higher diffraction power (more atoms or molecules coherently diffracts) which comes from more units across the physical size of the crystal. In protein crystallography we have much less units coherently scattering (by approximately 10 times in one dimension). Protein crystals are much less organized and much softer than small molecule crystals.

What you practically can do to improve your crystals is to apply all this knowledge. In order to remedy a sudden change in concentrations you should use a batch methods (larger volume crystallization), or solvent diffusion method (in a long plastic or glass tube with approximate diameter of 1mm, place protein on one side and precipitating solvent on the other side, or a membrane diffusion method (with protein in small volume contained with dialysis membrane submerged in precipitating solution). There are two varieties of the last method, capillary and the button. Finally, you can try to induce the crystallization through a very slow pH change.

In order to be successful though, you have to use a very reproducible high purity protein samples. However, you have to bear in mind that too pure protein is becoming difficult to crystallize so it would take longer to nucleate. Finally, you can try to use your regular crystallization conditions but replenish the protein part (place crystals in fresh crystallization conditions (some people call this macro-seeding even though this is technically incorrect).

As a final note, smaller crystals are also good for neutrons they just need more time per frame or larger flux. Good Luck.

Important thing is that what solvent you used for purification last time when you got the crystals. Just vary the concentration of pure compound in that solvent system, you will surely able to repeat them in a single crystalline form.

Hello,

I would definitely try seeding - macro and micro seeding. As far as i know neutron people grow large crystals by the macroseeding technique, but i have already seen large crystals growing after microseeding using small and quite irregular crystals.

Check the Acta D there are several reviews as well as (as long as i remember) a nice paper describing how to grow big crystals by the macroseeding technique.

Good luck

Hello ... I don't know about it, but I know a relevant research (GARCIA, Juan-Manuel) at Granada University & TRIANA lab (Spain) ... I'm sure that he is a good person to question about it. Good luck !!!