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Re: [ccp4bb] problem of crystallization
 

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CCP4bb <-- 2008 <-- May 2008 <-- 14 May 2008
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Subject: looking for software
From: Vellieux Frederic Frederic {- dot -} Vellieux {- at -} IBS {- dot -} FR
Date: 2008-05-14 		Next message:
Subject: Oxford cryostream 700 series to give away
From: Adriana Miele adriana {- dot -} miele {- at -} UNIROMA1 {- dot -} IT
Date: 2008-05-14


Subject: Re: problem of crystallization
From: Tom Walter walter {- at -} STRUBI {- dot -} OX {- dot -} AC {- dot -} UK
Date: 2008-05-14
A few ideas:

1) go even higher with protein concentration. Some ultra-soluble proteins may need >100 mg/mL. You may have to set up drops at higher ratios of protein:precipitant to achieve this. e.g. 3:1 or more. Use a test such as the Hampton PCT to tell you when you are in the right concentration range.

2) reductive methylation of lysine residues as Stephen suggested. This often reduces the solubility of proteins and will change the surface properties.

3) get rid of any glycosylation as the surface sugars increase solubility (and are flexible anyway and so bad for crystallization)

4) check the few drops which have precipitate and look for common factor e.g. >2M Ammonium Sulphate. You could then add a small amount of this ingredient (e.g. 100mM Ammonium Sulphate) to your protein and then re-screen.

5) Wait a bit longer. One week is still early days.

Good luck
Tom

** Tom Walter B.Sc. M.Res. **
** Oxford Protein Production Facility Tel: +44 (0)1865 287747 **
** Wellcome Trust Centre for Human Genetics Fax: +44 (0)1865 287547 **
** Roosevelt Drive walter@strubi.ox.ac.uk **
** Headington, Oxford OX3 7BN http://www.oppf.ox.ac.uk **


---- Original message ----
>Date: Tue, 13 May 2008 11:16:41 -0500
>From: Jennifer Han-Chun Tsai
>Subject: [ccp4bb] problem of crystallization
>To: CCP4BB@JISCMAIL.AC.UK
>
> Hi,
>
> This topic is not related to CCP4. I am having problem of
> crystallizing one protein. It's a pretty small protein with
> size around 15kDa. I have stock concentration around 100mg/mL.
> Crystallization plates I set up are with concentration of
> 10mg/mL, 30mg/mL and 50mg/mL. All the plates had been set up
> at least one week. Only around 5 wells per plate or less
> formed precipitation. The rest of wells are pretty clear
> still. Is there any suggestion for reducing protein solubility
> or increasing the chance of getting crystals?
>
> Thanks for your time,
> Jennifer

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CCP4bb <-- 2008 <-- May 2008 <-- 14 May 2008
Previous message:
Subject: looking for software
From: Vellieux Frederic Frederic {- dot -} Vellieux {- at -} IBS {- dot -} FR
Date: 2008-05-14 		Next message:
Subject: Oxford cryostream 700 series to give away
From: Adriana Miele adriana {- dot -} miele {- at -} UNIROMA1 {- dot -} IT
Date: 2008-05-14


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