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Two things worth doing are to make the following maps:
1. A CNS composite omit map. I crank the annealing temp up to 4000K.
Errors become very clear.
2. An EDEN map, preferably with about 20% perturbation, should also be
fairly unbiased.
Bill Scott
PS: In addition, you might want to idealize the geometry (skip the Xray
term) for a few cycles first. This seems to be important in Refmac
refinements. Then aim for a reasonably conservative geometry, with say
about 0.01 A rmsd bonds, since the resolution is low. I had a crystal
with 1 molecule per asymmetric unit with 80% solvent that diffracts to
3 A resolution, and the gap between Rfree and Rwork really was almost
10%. The main point of Rfree is to prevent over-fitting. If the Rfree
stops going down, continuing the refinement produces science fiction.
You might also want to try other methods of refinment, including TLS
instead of temp. factors, or torsion angle molecular dynamics rather
than cartesian, and so forth.
On Wed, 5 Jul 2006, Soisson, Stephen Michael wrote:
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Definitely agree with what Tim is saying below. Running refinement right
after MR can be very dangerous, actually giving you a much less informative
map. Discovered this the hard way on more than one occasion.
Steve
-----Original Message-----
From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Tim
Gruene
Sent: Wednesday, July 05, 2006 10:55 AM
To: Li Sheng
Cc: [email protected]
Subject: Re: [ccp4bb]: Re: Re: [ccp4bb]
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Did you refine the model after the molecular replacement? If so, do not do
so. Look at the maps directly and try to correct for any errors you see,
especially considering the difference maps (at 3 sigma).
If you run refinement without looking at the maps it would smoothen the
differences between the MR Model and your data and that is not desired at
this stage.
Tim
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
On Wed, 5 Jul 2006, [utf-8] Li Sheng wrote:
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Dear Tim Gruene,
This structure is a mutant of a protein previously solved. The space
groups for the wild type protein is P2(1)2(1)2(1) for one crystal and
C222(1) for the other. The conditions of crystallization for the two
proteins are different. Of course, this structure is solved by molecular
replacement. We have not added water molecules now.
We have tried both Rafmac and CNS, and the restraints was set to 0.05. But
we'll try again. Anyway, thank you all.
================= 2006-7-5 15:51, your message: Re:
[ccp4bb]:=================
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How did you create to this model? Was the gap between R and Rfree this
high from the very beginning? If not, try to find out where the gap became
so high.
- - did you introduce too many water molecules? You can overfit your data
this way by pretending noise were waters
- - do you have to tighten the weights of stereochemical restraints? If
you
use refmac5, try setting it to 'AUTO' or something below 0.1. A good
indicator off the weight being too high is the list of rmsd's at the end
out the refmac5 logfile.
Look at your difference map (fofc) at +/- 3 sigma, not 2 sigma. You want
that map to show as little density as possible (either 3 or 2 sigma, but 3
is more realistic, I dare say).
Tim
- --
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
On Wed, 5 Jul 2006, [gb2312] Li Sheng wrote:
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Hi, dear all,
Please do me a favour. I ever collected a data set of 2.7A. The space
group is C2. The model fit the 2fofc map and the omit map very well. The
R factor
is 0.26 now but R free is 0.37 and can not be minished. What kind of
error can cause such a gap between R factor and R free?
Thanks in advance.
Sincerely yours,
Li
2006.06.07
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.
============================================================================
================
Sincerely yours,
Li Sheng
2006-07-05
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