This was meant to Raji,
So here it goes to all.



---------- Forwarded message ----------
From: Zhang, Zhen <[log in to unmask]>
Date: Wed, Feb 22, 2012 at 12:15 PM
Subject: RE: [ccp4bb] Aggregated protein for crystallization
To: Pius Padayatti <[log in to unmask]>


Hi Pius,

I have done exactly that. I have one protein eluted at void volume of S200
column. The MALS experiment estimates more than 100 copies of monomer in the
aggregate. Against my belief, the protein crystallized and diffracted to 2.3A
and the resolution was improved to 1.6A later. It turns out that the
crystallization buffer breaks the aggregate to dimer and crystallized it from
there. I used the lower concentration of the crystallization buffer to run the
sizing column and the protein was eluted at reasonable elution time for dimer
even though the profile looks ugly and the purified protein is not
crystallizable any more. I guess the aggregate somehow protects the folding of
the protein and releases the protein slowly to the protein crystal in the right
buffer condition. So I think you should setup crystallization trials with your
aggregate protein. We cannot search hundreds of conditions for running sizing
column. So why not let crystallization trials find that for you?

Good luck.

Zhen

Marasco Laboratory
Cancer Immunology and AIDS
Dana Farber Cancer institute
http://www.marascolab.org/

-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Pius
Padayatti
Sent: Wednesday, February 22, 2012 11:55 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] Aggregated protein for crystallization

some more thoughts,
Do a cryo-EM imaging, it will be ideal than DLS.
if the particle sizes are uniform i would think your protein in that state
might be useful.
cheers
Padayatti

On Tue, Feb 21, 2012 at 6:21 PM, Raji Edayathumangalam
<[log in to unmask]> wrote:
> Hi Folks,
>
> As crazy as it sounds, if you have crystallized and managed to solve the
> structure of a protein from aggregated protein, please could you share your
> experience.
>
> After many constructs, many many expression schemes and after the usual
> rigmarole of optimization that is also often discussed on ccp4bb (buffers,
> glycerol, salt concentrations, pH, detergent, additives etc.), I now have a
> decently expressing truncated construct for my protein (80 kDa) that is pure
> but aggregated (elutes in the void volume from a Superdex200 column). I am
> tempted to make a boatload of aggregated protein and set up some crystal
> trays (after perhaps testing by CD). So I'd like to hear from folks who have
> been successful in solving structures from aggregates when many many known
> and tested optimization methods still leave one with aggregated protein.
>
> Thanks.
> Raji
>
> --
> Raji Edayathumangalam
> Instructor in Neurology, Harvard Medical School
> Research Associate, Brigham and Women's Hospital
> Visiting Research Scholar, Brandeis University
>
>



--
Pius S Padayatti,PhD,
Phone: 216-658-4528


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Pius S Padayatti,PhD,
Phone: 216-658-4528