Current Opinion in Crystallization

Current Opinion in Crystallization is a place where I deposit collections of opinions, tips, flames, personal insults, isolated case studies (better: reports) or anecdotal evidence on subjects where I (and not just I) lack definite and statistically valid indicators or probabilities to file it under knowledge. I would say that 90% of all crystallization tips fall into this category. 

All I am saying is that I take no responsibility for any damages of using the information given here, consequential, incidental, and otherwise. Follow-ups, in particular with hard facts, are welcome. Contributors are always asked whether they want their responses to appear on this public page.

1) Is Glycerol bad or good? (go to Summary)

Out of context, this is of course a stupid question, so here is my initial post:

Dear All :

I have a not crystallographic computing related but still interesting question. As often in protein crystallization, firm and validated information is rare and thus in this case I am happy to solicit also opinion and anecdotal evidence: Glycerol is used to protect proteins while being stored frozen. This is a particular issue for any high throughput operations, where the protein cannot be  processed immediately and needs to be stored in aliquots until machine time becomes available.

Dear Bernhard,

For four of the proteins/crystals that I have worked with using glycerol as a storage medium and in crystallization here are the stats:

3 proteins normally crystallize in 1 to 7 days without glycerol present in the protein buffer...i.e. they are removed from the buffer by desalting prior to setting up the trays. If 1% to 5% glycerol remains, crystallization of two proteins takes well over a month. The third never crystallizes.

1 protein absolutely needs glycerol for crystallization, without it you get crap. Conclusion from my experience, glycerol is more bad than good

A. Mesecar

I would guess that it will be hard to find this info, since most cases where
glycerol turned out to be harmful were probably never reported.  The
factoids are likely buried in some post-doc or graduate students memory
banks...Would be interested to hear if you turn anything up though!

Stephen M. Soisson

I have no hard evidence either, only anecdotal:

IMHO, glycerol doesn't really matter.  It does appear to make proteins more soluble, so if you have glycerol around one needs to use a higher protein concentration and/or higher precipitant concentration.  In  the CSHL macroxtal course, we have crystallized lysozyme with 20-25% glycerol (drops are 1:1 protein:well) in the reservoir for many years now.

I would not dialyze away glycerol unless extensive screening showed no results or poor crystals only.

We have used ethylene glycol in lieu of glycerol for lysozyme and that works. Ribonuclease crystallizes when ethylene glycol is used as the precipitant. I can't recall about ribonuclease and glycerol, though.

As for the response where glycerol had a negative correlation, I wonder if the experiments used the same conditions for +glycerol and -glycerol. From my experience, if you just add glycerol, without increasing the [protein] or [ppt], then you won't get crystals.

Jim

Hi "Hasta ls vista baby",

Try to read Cosenza, Bringaud, Baltz & Vellieux (2000), Acta Cryst. D56, 1688-1690.

In our case, glycerol was used as a crystal growth retardant, in order to slow crystal growth (thus avoiding the growth of non-diffracting crystals in seconds --- they diffract to 2.8A when grown in about 2 weeks --- we're busy preparing the manuscript describing the structure now).

So, sometimes there is no price to pay for the use of glycerol, you only reap the benefits!

Fred.

Servas,

Re: glycerol, here is my "mustard" (a lot of it you are certainly aware of):

1. We use glycerol to increase the solubility of proteins during purification 2. Storing proteins at -80 is generally bad (cold denaturation). Many proteins don't crystallize anymore after thawing, others, however, are fine. We generally try to avoid it. 

>Do I see this correctly that those which failed to crystallize after -8o did grow crystals before when not yet frozen?

Not quite. Some of the proteins that crystallized before freezing did no longer give crystals after storage at -80. On the other hand, I have never seen a protein crystallize after storage at -80 that didn't also crystallize before.

3. Qualitatively, we have noticed that glycerol, when used as a cryo-protectant, more often than not reduced diffraction quality. In most cases, that's compensated by the fact that one can now collect the heck out of those crystals. 4. Those proteins that required the presence of glycerol for solubility purposes crystallized just fine sooner or later.  I guess what I wanted to say was that we didn't really notice a correlation between using glycerol during purification and crystallizability. 5. Try screening the usual test proteins in the presence of varying amounts of glycerol and publish the results. Will Do.  6. I didn't think that crystallization setups are rate-limiting in high-throughput operations. 7. Instead of dialyzing, use centricons (or similar). In fact, after thawing, you will often find oligomers and aggregates in the sample. That's why we usually do a gel filtration run before crystallization (not quite suitable for high-throughput).

Mischa

Hello

maybe this article could be a good start.

Proc Natl Acad Sci U S A 2000 Jun 6;97(12):6277-81 Galkin O, Vekilov PG. Control of protein crystal nucleation around the metastable liquid-liquid phase boundary.

You can always lower the concentration of glycerol using PVA

Cryobiology 2000 May;40(3):228-36 Wowk B, Leitl E, Rasch CM, Mesbah-Karimi N, Harris SB, Fahy GM. Vitrification enhancement by synthetic ice blocking agents.

There will be no general rule: some proteins clearly donot mind while others clearly do. With dialysis, you will be on the save side.

Jeroen.

Hi Bernhard,

I have also been in this situation. I found that snap freezing gave good enough results without any cryoprotectant.

However, snap freezing must be carefully done. The frozen product must look fluffy and white, liked popcorn. The best way to do this is to squirt through a narrow gauge needle the solution into liquid nitrogen. Another way, for small volumes, is to put up to 50microlitres in an eppendorf, and toss the eppendorf into liquid nitrogen.

I also always thaw quickly, using my finger tips, so that the protein solution passes back through the freezing point as quickly as possible, and then I put the eppendorf on ice as soon as the solutions has completely measured.

You should compare identical experiments using fresh and freeze-thawed protein in parallel. I have no doubt that different proteins behave differently.

I observed no effect on diffraction due to the protein being freeze-thawed by this method, while glycerol did make a difference in the ability to grow crystals.

Anthony Duff

I routinely leave the glycerol in. 5% (which is what you get when you add 1:1 precipitant in a vapor diffusion) to 10% seems to have worked fine for me. More important, in my experience, is to greatly reduce the buffer strength so that ones efforts to change the pH of the crystallization solution are effective.

In any case, I crystallize with 1% glycerol, so that cryoprotection is easier.

Lisa

A better solution, in my opinion, is not to add glycerol at all: instead, concentrate it to something obscene, like 20 mg/ml, and freeze it in aliquots. Usually it acts as its own cryoprotectant at those concentrations.

This has the advantages that you have the identical protein available months later, and you don't have to do odious glycerol extraction with small volumes (which will also make aliquots differ).

Of course, I know there are exceptions, and not all proteins concentrate that high. (Sux.) But it's what I try first.

phx.

Hi,

Some proteins studied by NMR have shown large peaks for bound glycerol. These could not be removed by dialysis and glycerol had to be omitted from the prep.

An interesting reference regarding the benefits of co-solvents such as glycerol for stabilizing protein structure is: Stabilization of Protein Structure by Solvents, S.N. Timasheff and T. Arakawa, Chapter 14, pp 331-347 in Protein Structure a Practical Approach, ed T.E. Creighton, Oxford University Press 1990. This discusses the fact that co-solvents that have been found to stabilize protein structure are effectively excluded from the solvent region around the protein by preferential hydration of the protein. Proteins that show preferential binding of the co-solvent are generally destabilized.

For most proteins glycerol will stabilize protein structure and thereby improve the chances of crystallization. 

> not clear to me whether it is a conclusive extrapolation that stabilization equals better crystallization

For some however the opposite will be true.

Alun.

Summary on Is Glycerol bad or good? 

Probably more bad than good. If you don't need it, don't have it in. If you need it for stability, don't worry (actually, you are free to worry). There are no crystallization data on possible substitutes for glycerol either. 

Glycerol may be useful as a retardant when things grow too fast (problem also often seen in nanodrops?)

Snap freezing sounds interesting. Anyone else use that?

It would be a good idea to use the robots for a systematic study. Ok, I will. 

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