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: k.futte...@bham.ac.uk
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
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