Re: [ccp4bb] Crystallization with no precipitant

[D Bonsor]

Thanks for the all the comments on/off board. Especially for papers describing 
this method as it may be handy if the structure is solved. The crystals are 
reproducible. They have started to appear after 4 days. I can accelerate it to 
a day using just 1.5M NaCl as a reservoir solution, though I get excessive 
nucleation. 

Thanks again for the knowledge. 


Dan

Re: [ccp4bb] Crystallization with no precipitant

[Enrico Stura]

Dear ccp4bb,

1. Why would you need a precipitant to crystallize a protein?
The principle of salting-in/salting-out means that as you remove the salt  
that
keeps a protein soluble, the protein will tend to come out of solution and  
given

the right conditions, crystallize.

2. From the solubility curve as you increase protein concentration, you  
will reach a point at which
your protein will become supersaturated and possibly crystallize. You just  
evaporate the water.


3. Protein solubility is temperature dependent:
Huang M., Syed R., Stura E.A., Stone M.J., Stefanko R.S., Ruf W.,  
Edgington T.S. & Wilson I.A. (1998) The mechanism of an inhibitory  
antibody on TF-initiated blood coagulation revealed by the crystal  
structures of human tissue factor, Fab 5G9 and TF.5G9 complex. J Mol Biol.  
275:873-894.

Crystallized in the NMR tube when it was stored in the fridge
 De Halleux S., Stura E.A., VanderElst L., Carlier V., Jacquemin M. &  
Saint-Remy J.-M. (2006) Three-dimensional structure and IgE-binding  
properties of mature fully active Der p 1, a clinically relevant major  
allergen. J. Allergy Clin. Immunol.  11: 571-576.
The unglycosylated protein has low solubility - Concentration to 3.5 mg/ml  
and waiting for it to crystallize in the fridge.
Stura E.A., Visse R., Vera L., Cuniasse P., Dive V. & Nagase H. (2013)  
Crystals structure of full-length human collagenase 3 (MMP-13) with  
peptides in the active site defines exosites in the catalytic domain.  
FASEB J. 27:4395-4405.
A bridging peptide and low temperature induces crystallization - No  
precipitant.


4. A precipitant is an agent that is used by protein crystallogenists to  
avoid using too much protein
in the experiment. If salting-out does not work, PEG is used as a common  
crowding-out agent.

Ideally without a precipitant your protein will be in its most pure state.

5. Maïga A., Vera L., Marchetti C., Lorphelin A., Bellanger L., Mourier  
G., Servent D., Gilles N. & Stura E.A. (2013) Crystallization of  
recombinant Green mamba ρ-Da1a toxin during lyophilization procedure and  
its structure determination. Acta Cryst. F 69: 704-709

What about crystallization during lyophilization?

If you check throughout the literature, you  will finnd many more examples.

Enrico.

--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Proxima-2A, Soleil Synchrotron. Tel: 33 (0)1 69 35 8180 Beamline
http://scholar.google.com/citations?hl=en=Kvm06WIoPAsC=100=pubdate

Re: [ccp4bb] Crystallization with no precipitant

[CRAIG A BINGMAN]

On Mar 1, 2017, at 8:19 PM, Shaun Lott 
> wrote:

They diffracted, but weren't much use to us in the end, as they were hard to 
reproduce or optimise. Hopefully you have more luck than we did!

This sometimes happens with extremely soluble proteins.  If these are the only 
crystals you get, I might suggesting driving the protein concentration as high 
as possible (even to over 100 mg/mL in some cases) and then re-screening at 
substantially higher protein concentration than in the first pass.

Re: [ccp4bb] Crystallization with no precipitant

[Shaun Lott]

We have seen a similar thing once, where we obtained proteins crystals simply 
by dehydration of the drop:

Sharpe, M. L., Baker, E. N., and Lott, J. S. (2005) Crystallization of a 
protein using dehydration without a precipitant. Acta Crystallogr Sect F Struct 
Biol Cryst Commun 61, 565–568.

They diffracted, but weren't much use to us in the end, as they were hard to 
reproduce or optimise. Hopefully you have more luck than we did!

Re: [ccp4bb] Crystallization with no precipitant

[CRAIG A BINGMAN]

> Could it be possible that the robot never dispensed the reservoir solution 
> and the protein just crystallized against the reservoir solution through 
> simple concentration? I have just set up a couple of drops just against 
> reservoir solution. If this is the case, has anyone else experienced this and 
> had luck with this and got diffraction?

Yes.  Some proteins will crystallize in centrifugal concentrators.