Hi, You also can try the MR program Queen of Spades (Qs) ( http://utopia.duth.gr/~glykos/Qs.html; documentation, http://utopia.duth.gr/~glykos/pdf/Qs.pdf). It is a multi-dimensional MR, so it will use a lot of CPU time and memory, but might be worth try it. Good luck. Andrey Nascimento
2013/4/18 <[email protected]> > Stephen, > > Stephen, > > Although your peptide is smaller than the one I once worked on, here are > some thoughts that might be applicable. > > 1. Check and play with the radius of integration in the molecular > replacement. The default is probably not appropriate for your case but the > value should be much smaller than the default. > 2. Take a model - any model - of your peptide and make an arbitrary > artificial dataset by rotating the model around an arbitrary angle, and put > it in an arbitrary unit cell (say P1 for simplicity). Then use the original > non-rotated model and see what parameters will give you the best solution > and use those as starting parameters for your search. > 3. Consider that your model may be very asymmetric, i.e. much longer in > one direction than the other. In theory, you want the radius of integration > to be such that it covers one copy of the model but not more than one. If > the peptide is much longer than it is wide (which is somewhat likely), you > might run into the situation where the correct radius for the length would > incorporate multiple copies in the other direction(s). If this is the case, > I am not sure you can fix it. In my humble opinion, which might be very > out-of-date, this might be one of the reasons why MR does not work well on > small molecules. > 4. I think that it is possible that your crystal could be built from > multiple copies of randomly oriented copies of the peptide, which are > similar in their nature, but not exactly the same. This sounds odd but I > convinced myself a long time ago that such a crystal could be made. > > A long time ago I worked on a sea anemone toxin that had similar > properties. At the time I could not make step 2 above work, that is, I > convinced myself that I was unable to find parameters that did the job. > That was enough for my mentor to tell me that I should not pursue the > project... Of course such molecules are easily(*) resolved with NMR. > > Mark > (*) Relative statement, and not by me. > > > -----Original Message----- > From: Stephen Campbell <[email protected]> > To: CCP4BB <[email protected]> > Sent: Tue, Apr 16, 2013 7:39 pm > Subject: [ccp4bb] Difficult data > > 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 >
