From: Peter Hsu
Date: 21 September 2011 18:04
Hi all,
I've been trying to crystallize a 3 protein complex recently with little success. However, crystals of each subunit have previously been crystallized. I was wondering if any one knows of any literature/experiences where people have used seeds from an individual subunit to seed for a complex and succeeded? Or is this just a crazy/bad idea?
Thanks in advance for any input.
Peter
----------
From: Ed Pozharski
If there is one thing that I learned about crystallization, is that very
few ideas are so crazy that they are bad (i.e. not worth trying). Well,
if dried seaweed and ground horse hair are good for seeding, I don't see
how actual protein crystal seeds can be dismissed.
--
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs
----------
From: Peter Hsu
I forgot to mention, I can reconstitute the complex (co-expression/mixing proteins) and the complex comes off both ion exchange and an SD200 as one peak. Just haven't had luck with getting crystals.
----------
From: Enrico Stura
The idea is not at all crazy. In a sense it is quite similar to Stoichiometric variation screening* if you consider that the lattice of the crystallized subunit may contain planes
that might be conserved in the crystal of your hope for 3 protein complex.
*Stura, E.A., Graille, M., Taussig, M.J., Sutton, B.J. Gore, M.G., Silverman, G.J., Charbonnier, J.-B. (2001)
Crystallization of macromolecular complexes: Stoichiometric variation screening. J. Cryst. Growth 232:580-590.
Yet if the size discrepancy is quite large, the chances that will work will be quite slim.
Good luck,
Enrico.
-- Enrico A. Stura D.Phil. (Oxon) ,
Date: 21 September 2011 18:04
Hi all,
I've been trying to crystallize a 3 protein complex recently with little success. However, crystals of each subunit have previously been crystallized. I was wondering if any one knows of any literature/experiences where people have used seeds from an individual subunit to seed for a complex and succeeded? Or is this just a crazy/bad idea?
Thanks in advance for any input.
Peter
----------
From: Ed Pozharski
If there is one thing that I learned about crystallization, is that very
few ideas are so crazy that they are bad (i.e. not worth trying). Well,
if dried seaweed and ground horse hair are good for seeding, I don't see
how actual protein crystal seeds can be dismissed.
--
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs
----------
From: Peter Hsu
I forgot to mention, I can reconstitute the complex (co-expression/mixing proteins) and the complex comes off both ion exchange and an SD200 as one peak. Just haven't had luck with getting crystals.
----------
From: Enrico Stura
The idea is not at all crazy. In a sense it is quite similar to Stoichiometric variation screening* if you consider that the lattice of the crystallized subunit may contain planes
that might be conserved in the crystal of your hope for 3 protein complex.
*Stura, E.A., Graille, M., Taussig, M.J., Sutton, B.J. Gore, M.G., Silverman, G.J., Charbonnier, J.-B. (2001)
Crystallization of macromolecular complexes: Stoichiometric variation screening. J. Cryst. Growth 232:580-590.
Yet if the size discrepancy is quite large, the chances that will work will be quite slim.
Good luck,
Enrico.
----------
From: VAN RAAIJ , MARK JOHAN
agree, any crystallisation idea is worth pursuing, given you have or can make enough sample to try it with.
having said that, wouldn't you tend to select for the same crystals as the seed, i.e. crystals of the component on its own?
have you tried limited proteolysis of your sample, incl. a bit of protease in the drop - or can you think of ways to stabilise the complex?
Mark ----------
From: Patrick Shaw Stewart
This idea was discussed at the recent RAMC meeting, and there is at least one example where it has worked.
Generally, cross-seeding can work as long as you have homology. See e.g. Obmolova et al. Acta Crystallogr. 2010, D66, 927–933. The same group has reported seeding a complex with crystals of one of the monomers.
One thing to bear in mind is that there is no point in adding a seed stock (with e.g. crystals of one of the monomers) if the seed stock destabilizes your complex. This is all discussed in great detail and suggestions are made for finding alternatives in a paper that I mentioned here earlier (which we published this year) ref below.
Good luck
Patrick
_____________________
"Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization". Shaw Stewart et al, Crystal Growth and Design, 2011, 11 (8), p3432.
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