Dear Maruf,
I had a similar case, also to 2.4A. Those translations are often
caused by a non-crystallographic 2-fold rotation which is parallel to
the crystallographic 2-fold (there is an example in ruppweb as to why
that creates the translation and how to interpret it: http://
www.ruppweb.org/Xray/101index.html under "NCS with native Patterson
maps").
In your particular case, though, have you considered wether your
absences match the strong-weak pattern created by your non-
crystallographic translation? In that case maybe you don't have a 21
axis and your space group is P2 or C2.
If your space group is right, the non crystallographic rotation will
dominate your molecular replacement and your refinement and make it
difficult to spot good solutions from bad ones, since as long as MR
solutions recreate the non-crystallographic rotation (the axis, not
necessarily the correct position of your molecules around that
rotation axis), they will recreate the translation peak. I even got a
series of totally different MR solutions, all of them with beautiful
packing! For me it turned out that I had the correct MR solution for
months, but the refinement was stalling. Running refmac for 200
cycles (not less!) with very very tight geometric restraints
restraints produced a distribution of B-factors that pointed at which
regions were right (low Bs) and which wrong (high Bs). Removal of the
regions with high Bs followed by further refinement immediately
produced good maps, showing the new regions, and refinement proceeded
normally from there. Later on I could move back to normal weights,
too. Only after cropping the bad regions will the difference maps
really show anything (assuming the rest is right, of course).
Just based on one case, but your case is soooo similar to mine!
Good luck,
Jose Antonio Cuesta-Seijo.
**************************************
Jose Antonio Cuesta-Seijo
Cancer Genomics and Proteomics
Ontario Cancer Institute, UHN
MaRS TMDT Room 4-902M
101 College Street
M5G 1L7 Toronto, ON, Canada
Phone: (416)581-7544
Fax: (416)581-7562
email: [EMAIL PROTECTED]
**************************************
On Jul 14, 2008, at 8:42 AM, Maruf Ali wrote:
Dear all
I have recently collected several datasets on different crystals of
a particular protein with a resolution range form 2.4 - 3.2A. All
datasets seem to process well in p21 with a unit cell of 109.6
83.1 115.87 90 94.8 90, and this space group is further
supported by analysis with the program pointless. The dataset have
very reasonable statistics and Rmerge values, with no indication of
twinning. Analysis of the self patterson indicated a 43% off
origin peak at 0.3 0.5 0.47. This was further flagged by
pointless and molrep as a Psuedo cell translation (PST). Looking
at the systematic absences there are some unusually strong and weak
peaks. Initially after some toiling with molecular replacement,
there was a clear solution with four molecules in the asymmetric
unit. The maps generated were good enough to build the core of the
protein but do not look like maps generated from data at 2.4A-3.2.
Further more the free R is stuck at around 40%, and there is no
difference in free R when I apply ncs or not or any difference in
the maps. After building by hand and with phenix autobuild there is
still no difference in maps and Rfree. I have read papers where
labs have successfully refined PST data by separating the
reflections according to the PST. Oksanen et al 2006 acta D62
1369-1374: Poy et al 2001 NSMB Vol 8 no12 pg 1053: VajDos et al
protein science 1997 6;2297
My specific question are
Firstly how would I deal with refining PST data? (assuming this is
the problem).
Second with off origin peak of 0.3 0.5 0.47 how would I separate
the reflections?
Thirdly any comments would be valued
Thank you in advance
Maruf
Dr Maruf Ali
Section of Structural Biology,
Institute of Cancer Research,
237 Fulham road,
London.
SW3 6JB
The Institute of Cancer Research: Royal Cancer Hospital, a
charitable Company Limited by Guarantee, Registered in England
under Company No. 534147 with its Registered Office at 123 Old
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