Dear Zhihong,
Refinement stuck at 55% Rfree (which is essentially random), means that
you do not have found the correct MR solution. For me the prime suspect
is the space group. In my experience pseudo translation or any almost
crystallographic NCS will easily confuse automatic space group
determination programs like pointless and it is often trickey to find
out which symmetry is crystallographic and which is
non-crystallographic.
Since P21 is fairly low symmetry, I would reprocess your data in P1 and
just naively run all your favorite molecular replacement programs
(phaser, molrep, epmr, phenix, etc.) in P1. I would try them all since
there are subtle difference between these programs and one may succeed
where the others fail. Once you have a solution which refines (Free
Rfactor drops below say 40% in P1) you can try to figure out what the
true, higher symmetry space group may be. Your true space group may even
be P1, with pseudo P21 symmetry!
Good luck!
Herman
________________________________
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of xiaoyazi2008
Sent: Thursday, March 15, 2012 6:12 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Help! weird thing
Dear all,
Thank you very much for all the great suggestions on my case.
Yes, I run the latest version of Phaser in Phenix. The analysis
showed that there is one non-origin distinct peak more than 15 angstroms
from the origin.
44.1% origin: FRAC 0.000 0.042 0.500 (ORTH -15.7 2.8
103.5)
I managed to find four copies with the latest Phaser. After 50
cycles of rigid body refinement and 50 cycles of jelly body refinement,
Rfree/R goes around 55/52. It is really hard for me to do model building
at this point, because there is pretty much no new density.
Compare to model building and refinement with normal dataset (no
pseudo translation NCS), are there any special tricks or tips for
structure determination from dataset with pseudo translation?
Thanks again!
Have a nice evening or morning or afternoon!
Zhihong
On Mar 12, 2012, at 10:16 AM, Randy Read wrote:
Airlie points out that what I said about the ccp4i
interface wasn't correct! In order to keep the ccp4i interface in synch
with the version of Phaser, we've started distributing the ccp4i files
with the source code. The ones on our website are for an older version
of Phaser, but the latest ones will come with the Phenix download that
gives you the latest executable.
Apologies to anyone who was quick enough to download and
install the wrong ccp4i files already!
Best wishes,
Randy Read
On 12 Mar 2012, at 16:47, Randy Read wrote:
Yes, the current version of Phaser will do the
same test that xtriage carries out, and if it finds a sufficiently high
non-origin Patterson peak, it will automatically characterise the
translational NCS and use this for molecular replacement. This is
working pretty well in our tests.
In the near future you will be able to get the
current version of Phaser as part of the upcoming CCP4 release, but at
the moment the easiest way to get it is to download a recent version of
Phenix. You should be able to run that through ccp4i by downloading and
installing the updated GUI files from our website (and getting ccp4i to
interpret the command "phaser" as "phenix.phaser").
Best wishes,
Randy Read
On 12 Mar 2012, at 16:06, Anastassis Perrakis
wrote:
Hi -
I agree with Garib that its likely a
pseudo-translation issue.
I also agree with that the advice he
gives is correct, but ...
... since I am evidently less smart to
follow all these steps,
I like to use phenix.xtriage that will
tell me if there is pseudo-translation or not,
and will give a p-value for that being
significant. Its at the end of the text output.
I am not sure if Phaser deals these days
with pseudo-translation - I guess Randy can tell us.
If not, there is a very simple trick to
make Phaser work with pseudo-translation,
but since I threw the ball to Randy's
court and he told me the trick a few years ago,
I will let him explain only if needed
;-)
Best,
Tassos
On Mar 11, 2012, at 12:55, Garib N
Murshudov wrote:
Hi
Could you please check:
1) If there is psedotranslation. It
could be done by using sfcheck, molrep, ctruncate or calculating
patterson map and displaying using coot at 8-10 sigma level (it is my
favourite method for analysis of pseudo translations), whole unit cell (
a bit bigger than whole unit cell). Then if you see large no origin peak
(very likely along one of the axis, could be a). If yes then you have
several options: using phaser - 1) reduce cell, find solution in smaller
cell and then expand; 2) use molrep to solve. When there are two copies
related with pseudo translation molrep can give you solution; 3) as far
as I am aware latest version of phaser works with pseudo translation. If
you have pseudtranslation you should be aware that even if you solve the
structure starting R factors could be 70-80%. Then you may want to do 40
cycles of rigid body and 40-100 cycles of ljelly body
2) Check your space group in pdb and mtz
file. They may not be consistent.
I hope it helps.
Garib
On 11 Mar 2012, at 07:33, xiaoyazi2008
wrote:
Hi All,
I have an interesting thing to share.
2.3A dataset with good quality, P21
Partial model is available (~60% of the
target protein).
It seems that there are 4 copies in the
ASU (Matthews_coef 2.6, 53%solvent)
Molecular replacement gave two copies of
the model (Z scores are R6.2, T6.2, R6.8, T13.4). The solution is very
clear. It could not locate the rest two copies.
However, a quick refmac5 refinement gave
a very high R factor.
The funny part is the symmetry operation
in Coot.
As shown in the JPEG figure, it looks
like there should be another two copies (based on strong fo-fc green
map), which locate in the empty space between models found by Phaser.
Why is that Phaser could not find the
remaining two copies even there are strong fo-fc density?
Any suggestions...
Thanks a lot!
Zhihong
<weird thing.jpg>
Garib N Murshudov
Structural Studies Division
MRC Laboratory of Molecular Biology
Hills Road
Cambridge
CB2 0QH UK
Email: ga...@mrc-lmb.cam.ac.uk
<mailto:jen...@mrc-lmb.cam.ac.uk>
Web http://www.mrc-lmb.cam.ac.uk
<http://www.mrc-lmb.cam.ac.uk/>
P please don't print this e-mail unless
you really need to
Anastassis (Tassos) Perrakis, Principal
Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The
Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512
1954 Mobile / SMS: +31 6 28 597791
------
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: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.
www-structmed.cimr.cam.ac.uk
------
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:
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.
www-structmed.cimr.cam.ac.uk
