CCP4 bulletin board <[log in to unmask]> wrote on 05/14/2009 03:42:05
PM:
> Dear Crystallographers,
>
> I have exactly two spherulite crystals of a protein-peptide complex which
> have a fluorescently-labelled peptide in them, and are therefore nicely
> colorful in both the light and fluorescence microscopes, making it easier
to
> know that at least the peptide is in the crystal. However, they are not
> reproducible. Having gone through the usual list of possible variations
> which might account for the irreproducibility, I have hit the bottom of
the
> barrel. I was thinking that since the original crystals grew in utter
> darkness, undisturbed for two weeks while I was away, they were able to
> nucleate. Is it possible that light exciting the fluorophores is
detrimental
> to crystallization? Or perhaps the complete uniformity of temperature?
Even
> microseeding from one of the spherulites produced nothing (except in the
> original well.) Any brilliant suggestions welcome...
>
> Jacob Keller
>
Hi Jacob -
I just want to raise a warning, since a very similar situation bit me back
in grad school. I was trying to crystallize RecA with a
fluorescently-labeled oligo, and just like you, I got green-glowing
crystals, along with some glowing precipitate. I got extremely excited,
and a couple of months later had the structure done. There was no DNA in
the structure. I had crystallized unliganded RecA, and the fluorescent DNA
had precipitated all over the outside of the crystal, painting it and
making it look green!
Are you certain that your peptide doesn't precipitate under your
crystallization conditions? I hope for your sake that my problem is not
yours...
- Matt
--
Matthew Franklin , Ph.D.
Senior Scientist, ImClone Systems,
a wholly owned subsidiary of Eli Lilly & Company
180 Varick Street, 6th floor
New York, NY 10014
phone:(917)606-4116 fax:(212)645-2054
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