>> Have you tried DTT (or another way of keeping things in a reducing
>> environment)? This might help keep your disulfides (and hopefully
>> protein) happy.
>
>
> Surely the addition of a _reducing_ agent would _reduce_ your disulphides
> -
> i.e. break them.
Whoops...sorry, I guess I've got zin
In case that the poor diffraction quality is a result of problems with
flash cooling directly into liquid nitrogen, it may also be worth trying
to remove the cold gas layer above the liquid nitrogen prior to dipping
the crystal:
Warkentin et al.: "Hyperquenching for protein crystals", J. Appl.
On 24/01/07, Peter Adrian Meyer <[EMAIL PROTECTED]> wrote:
> We have crystallized a 21KD protein with 2 disulfide bonds grown for one
> month in 0.1M tri-sodium citrate pH 5.6, 0.5M (NH4)2SO4 and 1M Li2SO4.
The
> crystals look big (~0.4mm x 0.4mm x 0.3mm) and pretty (sharp edge, clean
> surface)
Hi,
You might want to look at
Berejnov, V., Husseini, N. S., Alsaiedc, O. A. & Thorne, R. E.
(2006). J. Appl. Cryst. 39, 244-251.
Regards,
John
John Rose Ph.D.
Associate Professor
B204B, The Fred C. Davison Life Sciences Complex
120 Green Street
Department of Biochemistry and Molecular Bio
-
From: "Juergen Bosch" <[EMAIL PROTECTED]>
To:
Sent: Wednesday, January 24, 2007 12:29 PM
Subject: Re: [ccp4bb] Fancy crystal, poor diffraction
Hi Tiancen,
nobody so far has suggested to mount the smallest crystals, 0.4x0.4x0.3
seems to be pretty large, this also means t
Hi Tiancen,
nobody so far has suggested to mount the smallest crystals, 0.4x0.4x0.3
seems to be pretty large, this also means that the freezing procedure is
longer. Then the other question is do you freeze in the Cryostream or by
plunging into LN2, I'd prefer the latter technique.
Good luck,
Jue
Tiancen,
I am not entirely clear - are you asking for techniques that can improve
your *existing* material - or are you shopping for *any* technique that
will eventually result in an improvement?
If you're looking for the latter, and are willing to send me your protein
sequence (I promise to keep
Tiancen,
One thing that you did not mention. Have you tried examining the
crystals at room temperature. Sometimes the cryo-cooling process, or
the cyro-buffer, can severely damage a crystal's long-range order. A
set of data, or just a few frames, collected at room temperature can
determine if
> We have crystallized a 21KD protein with 2 disulfide bonds grown for one
> month in 0.1M tri-sodium citrate pH 5.6, 0.5M (NH4)2SO4 and 1M Li2SO4. The
> crystals look big (~0.4mm x 0.4mm x 0.3mm) and pretty (sharp edge, clean
> surface) but diffracted to only 4A in-house. The spots are quite stron
Dear Tiancen,
for a survey of post-crystallization options, I highly recommend the following
review article:
Heras B, Martin JL. 2005
Post-crystallization treatments for improving diffraction quality of protein
crystals.
Acta Crystallogr D Biol Crystallogr. 61, 1173-80.
best of luck
Savvas
Qu
Roche Group: Chugai Pharmaceutical Co.,Ltd.
-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Tiancen Hu
Sent: Wednesday, January 24, 2007 6:54 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fancy crystal, poor diffraction
Dear all,
Sorry for the non-CCP4
Dear Tiancen,
I would suggest to try optimizing the hydration state of the crystal.
Either with a humidity controller (e.g. FMS system) at room temperature (or
4 degrees), with which you can optimize the diffraction,
or by partially drying the crystal on a filter paper soaked with some
precipit
7) Try different Cryo-protectants (can make a big difference)
8) Try to dehydrate the crystals by adding a low (5-10% w/v) concentration
of PEG20k or similar to the cryoprotectant prior to freezing
9) Anneal the crystals (a quick thaw/freeze - cover the cryostream nozzle
for ~5secs)
10) Grab the
Dear all,
Sorry for the non-CCP4 question. I think this is an old story but our knowledge
to deal with it is very limited. So any suggestions will be greatly appreciated.
We have crystallized a 21KD protein with 2 disulfide bonds grown for one month
in 0.1M tri-sodium citrate pH 5.6, 0.5M (NH4)
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