I have a protein that crystallizes between 10 and 16 month in high salt condition. Full length, no degradation. It is simply a flexible protein and needs more time to organize itself in a lattice. Nothing could be done about that so I made a lot of plates with different gradients, waited a year and picked all crystals.

The golden rule of protein crystallization is that there are no rules. Whatever gives you the crystal you need is legal. Just document it and you should be fine.

Proteins crystallize somewhere between a few minutes and never. I would never again throw out my screening plates unless they dry out.

and yes, shoot those crystals. There are many tests for salt crystals but I would always shoot first and ask questions later :)

Hi,

It is definitely possible to get crystals of your protein very late after setup of the plates.

Some people claim that they got crystals after a year or more. However, the likelyhood of salt crystals due to your plate drying out is probably at least as high. Easiest way to test is to put them on an in-house generate and look at the diffraciton pattern. If you get a decently sized unit cell you might have a winner.

This is not at all incommon. The salt is also rather benign. If you have enough crystals and they are large enough you may try to crush one. Salt crystal are muchh harder and may even produce a sound when they break. Hampton research also sells a stain you could try. Certainly, actually collecting a diffraction image should resolve this. If it is indeed a protein crystal you may be able to grow the crystals faster by increasing the initial protein concentration.

Getting crystals after a few months is common and could mean that you have to play with your crystallization conditions or sometimes what happens is your protein is getting degraded to a smaler, less flexible form that yields crystals. However, you have to check first if the crystals are not salt ( crushing, izit, running gel etc). If you have several crystals you could try any of the posibilities, but save a few in case you have to do microseeding.

Agreed, not uncommon at all. If these check out to be protein crystals I would check (i.e. via MALDI-MS) if there was some degradation of the protein and only the degraded form allowed for crystallization.

Little or no thinking required. Try to mount and get diffraction data. (for salt, there will be scatter even for tiny crystals). If possible, use the rest of the drop for some sort of seeding.

I remember a case where the crystal contained a truncated form of the protein. Some bits had to fall off first, and as soon as there was enough of it, the truncate crystallized. Crystals spotted after 9 months or so.

Also: don't ignore the buffer in your condition. It's doing much more than just buffering.

I have a protein that crystallizes between 10 and 16 month in high salt condition. Full length, no degradation. It is simply a flexible protein and needs more time to organize itself in a lattice. Nothing could be done about that so I made a lot of plates with different gradients, waited a year and picked all crystals.

The golden rule of protein crystallization is that there are no rules. Whatever gives you the crystal you need is legal. Just document it and you should be fine.

Proteins crystallize somewhere between a few minutes and never. I would never again throw out my screening plates unless they dry out.

and yes, shoot those crystals. There are many tests for salt crystals but I would always shoot first and ask questions later :)

Crystallization depends on kinetic circumstances - the way to get an equilibrium, finally by chance crystals. Of course, "long time" crystallization is frequently observed. Probably your crystallization can be improved (with respect to the time scale and crystal quality) by changing some parameter slightly.

Main question is, of course, do you have salt or diffracting protein crystals? Good luck!

If these are protein crystals, you can use them to seed another experiment and hopefully accelerate the crystallization process.

Hello,

I greatly appreciate and thank you for all the valuable suggestions and will surely implement them. It was amazing to hear some stories of crystal formation after some years.

Best,

Sagar

In the past I was working on a system that in the supersaturated Na+K phosphate

crystallised after 10-12 months. One of the way to survive was to use dialysis battons.

I still have couple of them around in scintillation counter glass containers waiting to be exposed :-) Benefit of keeping crystals for so many years (16 years actually) is that we potassium sights become occupied teaching us about interesting interactions of K with aromatic amino acid residues

Ho Leung Ng ·mentioned seeding (above). This is an important (and oft overlooked) step. Crystals need some sort of a "seed" on which they can begin to crystalize out form the solution. These "seeds" can either occur spontaneously or can be added to the crystalization drop with a horsehair, pin, or somesuch. Therese Bergfors and others have written some worthwhile papers on this subject

Sometimes a protein does not crystallize. But in the long run it can be modified, for instance by oxidation or proteolysis. The modified might be more prone to crystallize.

I have experienced getting protein crystals after 3 months.If wells are sealed tightly and there is no evaporation of mother liquor from the reservoir, It is not uncommon to get protein crystal.

Hi Sagar,

I do not know if it has been mentioned already but just in case, here are my two cents.

If you have enough crystals, run a SDS-Gel. Crystals appearing after long equilibration time could suffer cleavage (auto or endogenous) removing some flexible regions that impede the nucleation.

I think It's possible. I have this kind of experience, one of my proteins grew crystals after two months,and after I solved the structure to find there were almost 50 residues were missing at N-terminal. You may try to optimize the condition, may get crystals very soon.

Most cases, you can overlook a crystal trial after 2 months. However, I usually keep the crystal trial around for several years or until they are dehydrated. In my case, I worked with a yeast mating-type protein that would form crystals in some conditions in a month however they would only weakly diffract. Only crystals that grew over 4-6 months would give strong diffraction. Keep this in mind.

We had a case of an antibody scFv - The scFv by itself crystallized immediately, but if we added the hapten antigen, crystallization was delayed several weeks until the hapten in the solution had hydrolyzed. Once the antigen was gone, the scFv crystallized and formed the same crystals as the antibody fragment without hapten.

So very long lag times in crystallization may indicate that something has to change in your protein or other components of the solution before the crystals can form.

Thank you all for sharing your crystallization experiences. They're indeed valuable.

I got protein crystals after 1 year setting trials and the protein was intact . I even had a lot of wells with phase separation in the same plate and I got crystals also after such time. Every protein becomes a new experience and you never know. So, while the plates are sealed tightly and there is no evaporation of mother liquor from the reservoirs you shoud keep your trials, at least those with some initial signals, such as phase separation.

Our 'lab record' is 6 months. Beautiful clusters of orthorhombic plates appeared in a plate in the cold room that allowed solution structure. These crystals grace the Hampton research home page. Try them and see if they are any good.

I hope you've been lucky and found your crystals were protein and diffracted well. However I have to say that despite some other comments I would be surprised if you have crystallized the full-length protein. Crystallization is basically biochemistry - not magic - and your system would have come to equilibrium in hours or days unless it leaked slowly and became dehydrated. The typical reason proteins take a long time to crystallize is that some proteolysis is taking place and it is the proteolyic product that nucleates and grows crystals. A protease typically hits flexible regions and termini that may make poor crystal contacts. If your crystals were protein but you cannot repeat them more quickly or reliably, then I'd try adding tiny amounts of proteases to your drop. You can also try higher temperature and slightly wider screening conditions. Good luck with your crystallizations.

My record is 6 months. Forgot about a plate in the cold room. Somewhere between 4-6 months crystal rosettes formed and structure was published. Go figure.

"There is no theory that allows us to predict when or where proteins will crystallize."

This statement still holds true. In general you can still hope for crystals to appear until your solution is completely evaporated (this will eventually happen).

From "Protein Nanocrystallisation", Springer Series in Biophysics,

J.L.R. Arrondo and A. Alonso (eds), Advanced Techniques in Biophysics

Springer, 2006

I have had one that crystallized after several months on a shelf (and diffracted extremely well). Definitely the exception and not the rule, though. Bottom line though is the same whether a crystal grows after an hour or a month, while there a lots of things you can do to test whether it may be protein, you have to get it in front of an X-ray source and see if it diffracts.

If you're dying to answer the question about proteolysis before you are able to collect data (and you're willing to sacrifice a crystal), scoop out a crystal or two, dissolve it in gel-loading buffer and run SDS-PAGE. You'll know in an hour whether you have protein and if it is full length (NB. you may need to silver stain to get the sensitivity you want). If it turns out to be protein, you're one step closer. In this case, you'll probably want to use some of the crystals as seeds for later experiments so that you don't have to wait several months to get more crystals.

My longest growth time was 2 years and I was able to speed up the process to a few weeks by increasing the protein concentration in the repeat drop from 7mg/ml to 40mg/ml and also using a 5 :1 protein to precipitant ratio.