Hi,

If I were trying to solve the structure in subgroups of P22121, I would try to place multiple copies of a refined monomer rather than placing the tetramer.  I think that will give you a better chance of breaking the higher symmetry, if the crystal really has lower symmetry and twinning.

However, I agree that the statistics make twinning look less likely (but still worth keeping in the back of your mind).

One thing I noticed, looking at your Phaser log, is that the second pair of molecules has a B-factor about 15A^2 higher than the first pair.  It could be that this pair is significantly worse ordered (which would make your refinement more difficult), or it might even be an indication of some kind of statistical disorder.  It’s probably worth trying to figure out if there are, say, two different ways that the second pair of molecules can be placed.

The other thing I wondered about, looking at the Bdecay values in the aimless log, is whether you should try to reduce radiation damage.  If you had more crystals and another chance to collect data, you could try to do higher multiplicity data collection with lower exposure times, and then it would be easier to optimise a choice of where to cut off the merging of data.  However, radiation damage probably isn’t the main explanation for your difficulties in refinement.

Best wishes,

Randy

-----
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research    Tel: +44 1223 336500
Wellcome Trust/MRC Building                         Fax: +44 1223 336827
Hills Road                                                            E-mail: [log in to unmask]
Cambridge CB2 0XY, U.K.                               www-structmed.cimr.cam.ac.uk

> On 19 Sep 2015, at 12:47, Ben Porebski <[log in to unmask]> wrote:
> 
> Dear Randy,
> 
> Thank you for the suggestions.
> I've run my data through xtriage and it does not identify the presence of twinning, however it cannot be ruled out in the presence of pseudo translational NCS.
> 
> Log files are as follows:
> https://www.dropbox.com/s/jnspr3xby63x26i/3_aimless.log
> https://www.dropbox.com/s/61q2dplwj5xxhmr/4_phaser_MR.log
> 
> Looking at the tNCS/Twin table in phaser, it does not appear that the acentric second moment reduces.
> 
> When trying to solve the structure in P1, I used the original chainsawed model, and not the tetramer identified by phaser in P22121.
> I never found a solution in P1 with the chainsawed monomer, likely because of there being ~16 mols in the ASU, although the tetramer may assist this significantly.
> Interesting to hear about the latest version of phaser. I might give it a repeat with the latest phenix release.
> 
> Please take a look at the log files and let me know if you spot anything.
> I have also noted that there is a significant anomalous signal, which may arise from the Zn and As atoms in the crystallisation conditions.
> It may be possible to use these via experimental phasing to assist in structure determination, although I'm curious as to whether this is affecting regular MR.
> 
> 
> Thanks again,
> Ben
> 
> 
> On Sat, Sep 19, 2015 at 8:45 PM, Randy Read <[log in to unmask]> wrote:
> Dear Ben,
> 
> It looks like your solution is probably basically correct, but I think you’re right to consider that the true space group might have lower symmetry and you might have a combination of pseudosymmetry and twinning.
> 
> Is there any indication of twinning in the statistics?  The tNCS/Twin detection table in Phaser might be helpful.  If the crystal was twinned, you would expect an acentric second moment significantly less than two (after correcting for anisotropy and tNCS), although it wouldn’t necessarily be as low as 1.5 for perfect twinning, if it were pseudosymmetric (because the twin-related reflections are then already expected to be correlated before twinning).
> 
> When you tried to solve the structure in P1, did you use the original chainsaw model, or did you try the refined one with Rfree of 39%?  Although it’s a bad idea to pay too much attention to the statistics from an MR solution using a model refined against the same data, the refined model should give a stronger signal and might allow you to sort out any broken symmetry.  The true symmetry could also be monoclinic, with 3 different choices of which 2-fold is preserved.  It has recently become easier to test these options, with the latest version of Phaser (available in the current stable Phenix release, and coming soon for CCP4), because you can provide a subgroup as the space group, and Phaser will expand the data to the lower symmetry.
> 
> If you’d like to send me the logfile from the Phaser solution in orthorhombic (offline, to conserve bandwidth), I could take a look at it to see if there are any hints of other possible issues.
> 
> Good luck!
> 
> Randy Read
> 
> -----
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical Research    Tel: +44 1223 336500
> Wellcome Trust/MRC Building                         Fax: +44 1223 336827
> Hills Road                                                            E-mail: [log in to unmask]
> Cambridge CB2 0XY, U.K.                               www-structmed.cimr.cam.ac.uk
> 
> > On 19 Sep 2015, at 03:26, Ben Porebski <[log in to unmask]> wrote:
> >
> > Dear CCP4 community,
> >
> > I've been having trouble with a crystal dataset for the past couple of weeks and am completely stumped about how to best proceed.
> >
> > I have a small 12 kDa beta-sheet sandwich protein that crystallised in 0.2 M ZnAc, 0.1 M NaCac pH 6.5 and 10% 2-propanol. Through a stepwise increase of glycerol 10%-15%-20% and decreasing conc of 2-propanol, I was able to get diffraction to 2.2 Å at the MX1 beamline (Aust synchrotron).
> >
> > I have processed the data in mosflm, with a unit cell of 50.4, 71.4, 126.1, 90, 90, 90 and sg of P222.
> > Running through aimless and pointless suggests cutting the data to 2.4 Å and all other stats look reasonable (can supply if requested). Pointless guesses the sg to be P22121.
> >
> > Matthews suggests 4 mols in the ASU, and phaser identifies a translational NCS vector.
> > Peak Distance Vector
> > 22.3%  45.7Ã…:   FRAC +0.000 +0.417 +0.275   (ORTH    0.0   29.7   34.8)
> >
> > Searching for 4 mols in the ASU using a chainsawed structure (4U3H), where there are a handful of mutations only in the loop regions gives a single solution.
> >  SOLU SET RFZ=4.9 TFZ=9.5 +TNCS PAK=1 LLG=157 RFZ=3.1 TFZ=9.5 +TNCS PAK=2 LLG=113
> >     LLG=497
> >    SOLU SPAC P 2 21 21
> >
> > The solution looks like a reasonable tetrameric arrangement, however, problems arise during refinement.
> > Here, I am unable to have my R-free drop below 0.39. If I delete large sections of a single monomer and refine, I find that the electron density disappears.
> >
> > To me, this indicates that I do not have a correct solution.
> >
> > I have tried reprocessing in P1, but am unable to find a solution of the estimated 16 mols in the ASU.
> >
> > Going back to my diffraction images (attached), it seems that there are streaky spots throughout my dataset (even on multiple crystals) - so perhaps mosflm is having trouble correctly indexing.
> >
> > Any help or suggestions would be greatly appreciated.
> >
> >
> > Regards, Ben
> >
> > --
> > Ben Porebski,
> > PhD Candidate (A/Prof. Buckle Lab)
> > Department of Biochemistry and Molecular Biology,
> > Faculty of Medicine, Nursing and Health Sciences,
> > Building 77, Monash University, Clayton, Vic 3800, Australia
> > <xtal2_collect_1_001.jpg>
> 
> 
> 
> 
> -- 
> Ben Porebski,
> PhD Candidate (A/Prof. Buckle Lab)
> Department of Biochemistry and Molecular Biology,
> Faculty of Medicine, Nursing and Health Sciences,
> Building 77, Monash University, Clayton, Vic 3800, Australia