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Dear Stephen,
 
I guess you checked the processing that your huge unit cell is real. Did you try epmr? In this case an evolutionary search may be the most suitable method, but it could take a huge amount of cpu time.
 
Some comments:
Having about 40 monomers in the asymmetric unit could well explain the bad resolution you got.
In the old days, one had to specify a patterson radius for the rotation function, such as to get as many as possible intra-molecular vectors and as little as possible inter-molecular vectors. For large proteins, this is easy to achieve, for small peptides it is almost impossible because at a distance where there are enough intra-molecular vectors, there will also be many vectors from neighboring molecules. At that time, especially small but long helical proteins were extremely difficult to solve by MR. 
 
You may not be able to solve this MR problem and my advice would be: go back to the lab and try to get another crystal form.
 
Herman

From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Stephen Campbell
Sent: Wednesday, April 17, 2013 5:53 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] Difficult data

Hi Everybody,

Thanks so much for all the help!  I suppose I should give more info.  Marco, the data is twinned - I forgot about that in my initial posting.  I can't remember the stats, but it is not a perfect twin.  I thought that Phaser accounted for twinning, and I haven't got a solution good enough to worry about the twinning in refinement (so I haven't really been worrying about that at this point - maybe wrongly?).  I'll send out some diffraction images tomorrow if you would still like to see.

I'm fairly certain I'm working with the peptide - I have run SDS-PAGE as well as a Superdex-75 and the purity is perfect, and the size is what I would expect (3 kDa), so no obvious oligomers in solution. 

Dominica, the peptide is synthetic, but CD analysis suggests it is structured, and helical in nature.  It's is not cyclic,  but likely does have disulfide bridges keeping it fairly rigid.  The model I am using is an NMR model (sequence identity is almost 100%), but the ensemble shows very little flexibility, and any regions that show some sort of flexibility were trimmed (only two residues at each terminus).  I have also reduced the NMR model to one chain.  Are there other issues with the NMR models that I should be aware of?  I don't have much experience using them.

Pete, I did try all of the space groups within the P4 bravais lattice, but it didn't seem to matter which one I used.  Nico, that's what I thought too, but unfortunately I've never had such a large unit cell with such a small peptide, so I'm not sure at which point it gets ridiculous and there is more likely something wrong with the processing...  Going back to France, hey?  You're going to miss Edmonton soooo much!  Especially the warm winters.

Stephen


On Tue, Apr 16, 2013 at 9:28 PM, Stephen Campbell <[log in to unmask]> wrote:
Hello,

I am having a few issues with a data set I have been working on recently, and was hoping to get some ideas on how to deal with it, if anyone is in the mood.

I have been working with a very small bacterocin (about 3 kDa) and set up some crystal trays in hope of getting some high diffracting crystals.  I failed, but did manage to get a data set of reasonable quality to about 4A from crystals that reproduce very poorly.  Now, this sounds horrendous, but the MR model I have available is of a similar bacteriocin, whose structure is predicted to be essentially identical (different ORF, but almost identical sequence).  I was thinking it would be done in a day.  The bravais lattice is P4, and 118 x 118 x 165 (which seems HUGE for such a small protein...indeed calling it a protein is generous...peptide).  The data seems reasonably nice (nice spots, no visible overlap within 4A, but is very mosaic, about 1.5 degrees.

The data integrates and scales nicely, with very good chi2, R-factors and % rejected reflections.  It is hard to predict the correct space group, since all the tetragonal options have the same stats.  The systematic absences seem to predict p41212 (as does pointless), but it wouldn't be the first time I screwed that up.

I can't get a solution, no matter what I try.  Is this the nature of such a small peptide in such a large unit cell (placing the first model is difficult for MR since there may be many copies in the AU), or are there some tricks?  Is it likely that the unit cell is wrong?  Self Rotation functions give 8 peaks, but this is considering a peak fairly generously (approx. 15-20% of origin).  Are there some blatantly obvious red flags that I may be missing?  Any advice would be great - even if that advice is that it may be time to move on.

I should note that I have considered the idea that the peptide may be forming some sort of oligomeric structure such as a coiled coil, but it fails coiled coil predicting software, and there is no evidence that this should be the case.  The homologous structure is very rigid, and I would say fairly confidently that mine is likely the same - I just want to confirm it. 

Thanks so much,

Stephen Campbell
Post Doctoral Fellow
Department of Biochemistry
McGill University