Chen,

The first of your diffraction patterns looks streaky and I'm  
wondering whether your intensities carry a significant error due to  
poor peak profiles. I know you used already two alternative data  
processing programs, but I'd recommend to give XDS a try. When  
dealing with poor peak profiles (for a subset of frames) I got  
distinctly better results with the latter. You may need to start the  
integration with frames that show good peak profiles. (I had to  
renumber frames to get XDS to properly integrate the more streaky ones).

As XDS will integrate in P1, unless you choose otherwise, you can  
easily test merging stats in a systematic fashion. If your true  
symmetry group may be lower than P622, then it is definitely  
worthwhile to try lower symmetry when running phaser.

Klaus


=======================================================================

                     Klaus Fütterer, Ph.D.
                 Reader in Structural Biology

School of Biosciences		  P: +44-(0)-121-414 5895
University of Birmingham	  F: +44-(0)-121-414 5925
Edgbaston                         E: [log in to unmask]
Birmingham, B15 2TT, UK           W: www.biochemistry.bham.ac.uk/klaus/
=======================================================================





On 30 Jun 2010, at 05:50, Chen Guttman wrote:

> Dear Colleagues,
> Im on the bring of giving up on a very difficult and puzzling  
> structure and my last hope lies here with you folks...
>
> Background:
> Im working on a point mutated protein with a known w.t. structure.  
> This specific mutation have been crystallized in the form of plates  
> (C2221 SG) and hexagons (P622 SG). Whilst the plate form structure  
> was solved without of a hiccup, the later was a different story. I  
> am attaching two images of the obtained diffractions of the hexagon  
> crystal, 90 degrees apart (the 905 images were collected at a  
> synchrotron beamline with a delta phi of 0.2).
>
> What I've done so far:
> Diffracted images were indexed & integrated with iMosflm (used  
> pointless to asses the correct SG). This was later scaled in SCALA  
> to 2.4A with the attached statistics.
> HKL2000 was also used but it couldn't lock on the P622 symmetry  
> (gave either C2 or P2 which fit to the first section of the data  
> set but didn't fit the 90 degree diffraction pattern); Splitting  
> the data into two data sets at the 90degree gave a P6 option but  
> once it finished the first 90 degree, it missed-fitting the 2nd  
> data set.
> Scaled data was phased using Phaser with the known w.t. structure  
> which gave a single solution with the following statistics:
>  RFZ=7.6 TFZ=33.4 PAK=0 LLG=1316 LLG=1426 and with an initial R- 
> factor of 48.7
> Looking at density map, I could distinguish areas which had  
> excellent fit while other areas demonstrated poor fit. The ligand,  
> which was soaked with the crystal and was also solved with the w.t.  
> protein, fitted almost perfectly with the identified positive  
> peaks. However, apparently there were also major changes (mainly  
> positive peaks) that could not be accounted for by the structural  
> restraints and were hard to figure out. These major changes, which  
> might as well be ghosts of a problematic data set, are the reason I  
> want to solve this structure
> Rigid body, Restrained refinement and real space refinement didn't  
> improve Rwork/Rfree beyond 0.4/0.46.
>
> I've also tried the following steps:
> Used AMore and Molrep - didn't yield any improvement in density map  
> nor in the R factor statistics
> Used Phenix Refine with different settings including simulated  
> annealing, didn't work either
> Tried rebuilding using Autosol and generation of OMIT map - this  
> yielded some sort of improvement to 0.39/0.44 but still the map was  
> not drastically improved and there where areas which were hard to  
> fit with the current model.
> Tried removing stretches of residues and check the map after  
> refinement - mostly i've noticed spots of positive peaks related to  
> the backbone.
> I've also checked for wrong use of symmetry with several programs,  
> including Zanuda, and still the best SG is P622.
> I've checked for twining with Phenix, CCP4 and web hosted programs  
> and no twining was detected (not even perfect twining).
> So, I've pretty much hit a brick wall. I would be happy to hear any  
> suggestion to what might be the problem with this Data set.
>
> Thanks for the help and time,
> Chen
>
>
>
> -- 
> Chen Guttman
> The Zarivach laboratory, Building 39, Room 009B
> Ben-Gurion University of the Negev
> POBox 653
> Zip Code 84105
> Beer-Sheva
> Israel
> http://lifeserv.bgu.ac.il/wb/zarivach
> Tel. +972-8-6479519
> Fax. +972-8-6472970
> <45deg.jpg><135deg.jpg><Statistics.log>