Query 1 - I wonder why your Rfree is so unreasonably close to the
working R? with data to 2.4A I would expect it to be much more
different. One reason why this can happen is when the NCS is reflected
in the diffraction.
Do you have more than 1 molecule in the asymmetric unit, and could you
then have higher symmetry? I note a is approx. equal to b - could you
have a "non-crystallographic" pseudo 4 fold? ( Look at an hklview plot
of hk0 hk1 etc, and see if there is any indication of such symmetry..)
If that were the case, and all hkl was approx equal to k-h l then Rfree
would not really be "free"..
Comment 2. In my experience brocedures after MR depend on the quality of
the original model. First as Tassos says, the MR programs all do a
pretty good job of rigid body refinement at the resolution you specify,
so there isnt much benefit in repeating it, unless you can break the
solution up into different domains. Phase extension can help espec if
you can average molecules, but when your first R factor is so good, I
wouldnt think it was necessary.
Comment 3 - The difference between Rfree of 23% and 24% is pretty
neglibible - both are much lower than expected at 2.4A
Eleanor
Anastassis Perrakis wrote:
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On Sep 9, 2006, at 3:20, Qun Wan wrote:
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Hi,
I have a couple of questions during model refinement. Hopefully
someone could help me.
My crystal diffracted to 2.4Å resolution with molecular weight 40KD.
The crystal belongs to P212121 space group with unit cell dimension
a=72Å, b=73Å, c=88Å. The crystal is not twinned. After molecular
replacement in the resolution range 20-4Å, I got a very good peak
for later CNS refinement.
In rigid body refinement, I am not sure which resolution range I
should use, 20-4Å or 20-2.4Å? I tried both. If I use 20-4Å data,
R=0.3355, Rfree=0.3265. If I use 20-2.4Å data, R=0.3304,
Rfree=0.3207. After 20-4Å rigid body refinement, I also do phase
extension gradually from 4Å to 2.4Å (that is, rigid body refinement
20-4.0Å, then 3.5Å, then 3.2Å, ...until 2.4Å), the result is
R=0.3360, Rfree=0.3268. From the results of R and Rfree, I believe I
should use 20-2.4Å in rigid body refinement. However, I was told to
use 20-4.0Å first, then do phase extension like I said above. Could
anyone tell me why?
I don't know why you were told to do so. Maybe the person who had
told you to do so, can tell you why he told you to do so, so you can
tell us why he told you to do so ? ;-)
I would use 20-2.4, as you concluded by yourself - in my opinion
correctly - that it is the best thing to do.
I would even say that molrep/amore/phaser do good enough rigid body
refinement, that you don't need to do it at all in most cases, and
you can jump straight to refinement (of course, ARP/wARP+REFMAC would
do a good job at 2.4 A and give a good map with a fairly complete new
model and would have minimized manual intervention - try it next time
...!)
The second question is after final refinement and model building, my
R=0.2133, Rfree=0.2333. It looks good. Then I validate the model by
some other programs, like procheck, what If, and MolProbity. After a
painstaking work, I am frustrated to find that my R=0.2056,
Rfree=0.2398. I am confused why after rotamer correction,
Ramachandran check, water check, collision check, etc., my Rfree
increased, not decrease?!
Well - no answer, but I would not worry about it. The difference is
not really significant at all.
But, your model is significantly better after correcting all the
details - well done !
btw: R and Rfree are a bit too close - might be due to your over-
careful work ;-)
But both numbers indicate you did a good job - so enjoy the structure.
Tassos
Your help is greatly appreciated!
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