Re: [ccp4bb] Protein-DNA complex for crystallization

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Re: Protein-DNA complex for crystallization
From: "Das, Debanu" debanu {- at -} SLAC {- dot -} STANFORD {- dot -} EDU
Date: 2007-07-16

Hi,
You can try the following to improve your protein-DNA complex crystals:

1) Try HPLC purified oligos. You can get that from IDTDNA instead of the standard desalting form. Alternatively, if you have access to a reverse phase HPLC/column, if you order DNA with Trityl group on, run it on the HPLC, take trityl group off and run a second time to get pure DNA. But these days with the advancement of DNA synthesis methods, I think there there's not much of partial/incomplete product contaminating the samples.

2) Definitely try seeding under different protein concentration, protein:DNA ratios, etc.

3) Have you tried different protein:DNA ratios? That can have a significant impact for complex crystals. Also try modifying the DNA ends, overhangs,etc. You can also try different DNA lengths, design them so that they overlap end-to-end to complete helical turns,etc.
Refer to:
Tan, S. , et. al JMB 2000,297(4), 947-59.

Thanks,
Debanu.

________________________________

From: CCP4 bulletin board on behalf of bputcha
Sent: Mon 7/16/2007 9:01 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein-DNA complex for crystallization

Hi,
I am trying to crystallize a protein-DNA complex. I purify the protein finally
using gel filtration. I purchase
single stranded complementary oligos (desalting from idtdna.com), mix them up
and make DNA duplex by
heating to 95 degree C and cooling to room temperature. I mix protein and DNA,
concentrate and use it
for crystallization.
I am geting small crystals consistently under a specific condition. These
crystals take up IZIT dye but are
not well shaped. I am not able to improve the size and shape of the crystals
substantially even after
screening with additives (Hampton research).
I suspect that purity of the duplex DNA (presence of unpaired oligos) is
limiting the chances of obtaining
better crystals.

How can I purify the duplex DNA further?

Are there better ways of making protein-DNA complex for crystallization?

If I make the protein -DNA complex and then do the gelfiltration, will the
complex purified so be a better
choice for crystallization?

Thank you
Kumar

Dept. of Biochemistry, Cellular and Molecular Biology,
Walters Life Science, # 406,
University of Tennessee, TN, Knoxville, USA

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Scientist Position Available
From: Christian Wiesmann wiesmann {- dot -} christian {- at -} GENE {- dot -} COM
Date: 2007-07-16

Next message:
Subject: Re: about Procheck
From: Ethan Merritt merritt {- at -} U {- dot -} WASHINGTON {- dot -} EDU
Date: 2007-07-16