Disappearing crystals

Hi All,

I have just noticed a very strange thing and need some help in understanding it.  I recently found two crystals in a condition from a screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and 30% PEG MME 550).  The small crystals appeared after a month and started to grow over the next 5 days after I first saw them (see pictures attached).  I just check the same drop today and now the crystals are gone.  So I was wondering what happened and if anyone experienced this before.  Any insight or advice on what to do would be greatly appreciated.

Thanks

Christine

From: David Schuller

Was the heating/cooling turned down over the Turkey day holiday?

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From: YoungJin 

Hi Christine,
I had similar experience. In my case, another crystal showed again with different size a few days later. Sometimes, it seems like it is a common event to others as well as I heard although my case only takes about a week to be crystallized.  I'd rather wait or just set up again or in a slightly different way.

Wish you well.

Young-Jin

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From: Enrico Stura 
When advice on crystallization is needed, it is important to give details of
the protein concentration, the buffer the protein is in as well as the method
used to grow the crystals.

Problem: The crystallization conditions are essentially low salt: 100mM buffer
and only 50mM CaCl2. So the buffer that the protein is in is very important !!
Fluctuation in the reservoir/drop environment will lead to crystals dissolving.

Solution: Balance the salt in the reservoir and in the protein:precipitant drop and make sure
the temperature is kept constant.

Since I do not have all the necessary information, the diagnosis and the solution proposed
are likely to be wrong!

Enrico.

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From: Jan Dohnalek 
We have seen such behaviour connected to temperature fluctuations.

Jan

--
Jan Dohnalek, Ph.D
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From: Fischmann, Thierry

Yes, and if they don't reappear it may be worth moving the trail to an incubator set at a slightly different temperature than "room temperature". You may have to try both lower or higher temperatures if you have no idea how the temperature may have changed (it's the temperature of the room when the crystals showed up that matters, obviously).

Alternatively you could set up "fresh" trails with same conditions but for a deliberate and controlled screening around what the room temperature typically is.

Good luck

Thierry

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From: Harman, Christine 
Hi,
Thank you so much for your replies.  A lot of you have mentioned fluctuations in temp as the major contributor.  And a few of you have asked for more details of my protein/buffer and set up.  To my knowledge, the tray has been kept at constant 20 C (in an incubator) with exception of course to when I remove the tray to view the drops.  It could be possible that my inspection of the tray might have contributed to an increase in temp, but only temporarily.  I am very careful about the time the drop sees intense light, but it is possible the temp could have changed enough to cause this problem.  Just to give a few more details.  My protein (a Fab fragment/peptide complex (hopefully) is in buffer containing 100mM Sodium Acetate pH 5.5 with 150mM NaCl at a protein concentration of ~4.3mg/mL.  After setting up my drop with reservoir solution I add NaCl to well to give ~75mM NaCl to match ionic strength of protein in drop which is diluted 1:1 with well solution.  I do hope this problem is temperature.  Although I am a little sad to not be able to freeze those crystals I did see, I still consider myself lucky to get such good result from a condition right from the screen so there will be some definite optimization set ups with this condition.  Could I safely say though that the crystals I observed are not salt..:)  I guess that is one good thing to take from this.  Any more suggestions on optimization would be very welcomed.

Thanks again to all you,

Christine

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From: Mathews, Irimpan I. 
Hi Christine,

Another minor point (mainly for hanging drops) not mentioned in the messages.

When you bring the crystallization tray from lower temperature to room temp. there is a possibility for condensation and slowly droplets near the protein drops gets absorbed into the protein drop.  If this happens, depending on the condition and character of the protein,  the crystals could crack or dissolve.

In your case, condensation might have happened during your last check and the crystal may have affected by this issue.  To avoid this problem: while taking the tray from the incubator place another tray on the top of your protein tray and keep them outside for few mts before checking with the microscope.

best wishes,
Mathews

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From: Artem Evdokimov 

In addition to others' excellent suggestions, I'd like to offer more option to consider:

Your crystallization set-up has some form of chemical non-equilibrium with respect to protein. It may just be my imagination but something is happening to the yellow precipitate in your drops, namely it's going away. Is it possible that you have a slow proteolysis (or something) happening to your protein, where one of the proteolytic products is crystallizing, however it's not the *final* proteolytic product so crystals are transient for as long as a 'steady state pool' of the appropriate form is above the solubility threshold (this implies equilibrium between soluble protein and crystals, which is not a huge stretch).

If this is the case, I'd check your drops with MS after a few days to see if anything interesting is happening. Options include but are not limited to: proteolysis, oxidation, chemical reaction of protein, chemical reaction of buffers and/or additives, and so on :)

Artem

P.S. It could simply be that your protein is initially precipitated but then driven back into solution? It happens.

A.

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From: Enrico Stura 
Dear Christine,

I had guessed that you had more salt in the protein solution
than in the reservoir with a relatively  low protein concentration.

The protein is in:  and the reservoir is: We can suspect that no matter what temperature the equilibrium will move
towards dilution in the drop.

Basic suggestion:
Set up a range from 20-30% PEG MME 550
with at least 150mM NaCl in the reservoir.

Fine tuning:
Add high MW PEG (10K or 20K 1-5%) it should have a stabilizing effect
Try a range of salts apart from NaCl: The clasics are: Li2SO4, KSCN, MgCl2.
Try additive amounts od MPD 0.5-3%.
Try a small tight range of various pH.

Enrico.

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From: Patrick Shaw Stewart 

Christine, I have a couple more comments

First, if those crystals come back, I would certainly try microseeding into random screens.  You should be able to pick up new conditions that have a little more salt in them, and are therefore stable regarding the movement of water.  (If you can't get the crystals back, I would try microseeding with the precipitate that you have.)

Second, it's helpful to understand that the movement of water in and out of the drop is connected to heat flows, not temperature.  The heat flow drives the movement of moisture (in addition to the salt etc. of course).

Once you understand that you can easily get rid of e.g. condensation on the tape by putting e.g. a warm book on top of the plate in the cold room (or a book from the cold room under the plate at room temp).  Similarly, you can prevent dilution of your drops by thinking about heat flows.

Some say that it's essential to use Blundell and Johnson.  Others say only Bergfors on crystallization, Lord of the Rings or even Harry Potter can provide the necessary inspiration.

Good luck

Patrick

Ref for microseeding: Allan D’Arcy, Frederic Villarda, May Marsh. An automated microseed matrix-screening method for protein crystallization.  Acta Crystallographica section D 63 (2007), 550–554.