[ccp4bb] Purity of DNA oligos in protein co-crystal attempts.

[Charles Allerston]

Dear all,


I am trying to decide on the many variables I need to consider to attempt a 
co-crystal between a protein I work on and a DNA oligo of ~35nt in length.

As well as molar ratio which from the literature I have seen 1.2-1.5 in excess 
of DNA, I note from the various oligo companies that there are several levels 
of purity available.

I would be very interested to hear from you about your experiences specifically 
trying VARIOUS purity levels and also the specific company you used?  Also, any 
rationalisation as to what the contaminating entities are which may prevent 
co-crystallisation would be welcome.

If you reply direct to me, I would be happy to provide a digest of responses 
for the board?

Any help/advice/anecdotes would be most appreciated.

Cheers,

charlie



Dr. Charles Allerston
Genome Integrity Group
Structural Genomics Consortium
Nuffield Department of Medicine
Old Road Campus
University of Oxford
OX3 7DQ
http://www.sgc.ox.ac.uk/

Re: [ccp4bb] Purity of DNA oligos in protein co-crystal attempts.

[Ed Pozharski]

On Wed, 2011-07-27 at 13:42 +0100, Charles Allerston wrote:
 I am trying to decide on the many variables I need to consider to
 attempt a co-crystal between a protein I work on and a DNA oligo of
 ~35nt in length.  
 

In my somewhat limited protein:DNA crystallization experience, the less
pure oligos are just fine.  I suspect that protein:DNA complex
crystallization is just like protein crystallization - every system is
different and whatever worked for someone in the past may not be
applicable in your case.  Basic rules are (may the protein:DNA
crystallization experts correct me and expand the list):

1.  Keep the DNA length close to whole number of turns.
2.  Try blunt ends, single and double overhangs.
3.  Slight DNA excess.
4.  May try less purified (and thus cheaper) DNA first.

Some anecdotes:

1.  Everyone knows complex can be purified, but it is rarely done.
2.  Screens designed to crystallize protein:DNA complexes are a hoax.
3.  Bet on PEG!
4.  Don't give up on 10A resolution - just shoot ~1000 crystals and one
will give you a nasty twinned 3A dataset.

Cheers,

Ed. 


-- 
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs