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<div class="moz-cite-prefix">Hi,<br>
<br>
just for completeness... Correctly selecting free-r flags is
essential in case of twinning (phenix.refine does it by default).
If flags were chosen without having that in mind then "R=0.24,
Rfree=0.28" from your example may easily become "R=0.24,
Rfree=0.34" once you switch to correctly generated flags. I've
seen it more than once.<br>
<br>
Pavel<br>
<br>
On 5/31/13 12:15 PM, Jon Schuermann wrote:<br>
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<div class="moz-cite-prefix">Sorry if this is a little long, but I
saw that more than one person was having a similar problem.<br>
<br>
First, I agree with everything Nat has said, but I will explain
a little bit more. Twinning and/or pseudosymmetry can be a hairy
issue to deal with. In some cases, NCS can be a real pain. I
have seen many cases where just pseudosymmetry is present, but
people assume it is twinned as well. To be fair, it is very easy
to come to that conclusion, especially at the early stages. Let
me just set the stage with a hypothetical example... <br>
<br>
You collect a 2.5A dataset that processes well in P622 with an
Rsym=0.11 (overall). The intensity statistics look normal, which
I won't go into right now because it is complicated (even though
Nat eluded to it with the 'L-test'). You perform MR with a
search model that has 60% sequence identity and get a solution
with 1 molecule in the AU in P6522. The solvent content is 0.6.
You run the solution through rigid-body, followed by positional
and B refinement with simulated annealing. The resulting R=0.35,
Rfree= 0.40. You look at the maps and the density does not look
good. You can see density for most of the main-chain but almost
all of the side-chain density is not present. You go about
trying to build in the density but the R/Rfree do not drop much.
What is your next step? Some would go to Se-Met or heavy atom
soaks to solve by SAD or MIR, but the results are the same...
Now What? Most people would say, its hexagonal where merohedral
twinning is possible so it must be twinned. You rescale the data
in P6, P312, P321, and even P3 (tetartohedral twinning) and the
Rsym is about the same in all the subgroups with P6 and P3 being
the lowest with Rsym=0.08. You perform MR in P65 and get a
solution with 2 molecules in the AU. To 'check' if it is
twinned, you run refinement twice from the same starting model;
first, twin refinement specifying the 'twin law' (from XTRIAGE),
and second regular refinement and then compare the results. You
think the results speak for themselves... with the twin
refinement, the R=0.24, Rfree=0.28 with the twin fraction
refining to 0.49 (nearly perfect twinning). The maps look great!
The regular refinement (without specifying the 'twin law')
refines to R=0.30, R-free=0.32 and the maps look better than
P6522, but not as good as the maps from the twin refinement. You
think, 'This has to be twinned!' But here is the kicker, IT'S
NOT TWINNED!!! How could this be???<br>
<br>
When you input the 'twin law' in refinement and the twin
fraction refines to >0.4, Phenix detwins using the
proportionality rules. This method uses the model in the
detwinning process which WILL introduce model bias in the maps,
so they look great. Even if your model is NOT correct, the maps
will still look good, because of the model bias. For this
reason, you should calculate several 'omit maps' over different
regions of your structure and inspect them, or look at maps
calculated from refinement when you did NOT specify the 'twin
law'. As for the R-factor being lower, the calculated twin
R-factor is usually lower than the standard R-factor since the
equations are NOT the same. So, a drop in R-factor at this early
stage is not necessarily conclusive that your data is twinned.
There are a couple of papers explaining this (which Nat eluded
to and I believe I reference below).<br>
<br>
I have seen many people get 'itchy finger' and try to use the
twin refinement too early. As Nat mentioned, you don't want to
run it unless you are at the end of model building and ready for
deposition, but the R-factor is still too high. Then it may be
necessary to run refinement with the 'twin law' included to see
if the R-factor drops 'significantly'. <br>
<br>
To summarize, if you suspect an issue with twinning and/or
pseudosymmetry since the maps don't look as good as they should
for a given resolution and the R-factor is too high,
rescale/reprocess your data in lower symmetry SG's and rerun MR.
When you get a solution, refine it WITHOUT specifying 'twin law'
and look at the maps. <br>
A)If they look much better than the solution in the higher
symmetry SG, continue your model building and refinement
(WITHOUT 'twin law') cycles in this lower symmetry SG until the
model building is complete and you would be ready to deposit the
structure in the PDB. If the R-factor is still too high, then
run refinement including the 'twin law' with the twin operator
from Xtriage. If you see an appreciable decrease in R-factor,
then you probably have twinning. If the R-factor dropped, but is
still not low enough, then either your model is still not
completely correct or you have something else going on as well
(see end of B below).<br>
B) If the maps still do not look any better than the higher
symmetry SG, try MR in another lower symmetry SG and repeat. I
sometimes go down to P1, if I have enough data. If the maps
still look bad (assuming you have a complete dataset and MR
solution), then you may not have a SG issue at all, but
something else. I would look into other issues including
anisotropy, pseudo-translational symmetry, order-disorder,
missing molecules, etc. I will usually scroll through the images
to see if I see something obvious. Reprocess the data more
carefully looking for things like extra spots not predicted, or
predicted spots that aren't actually there, etc.<br>
<br>
Here are a few references to papers that explain some of these
issues more in depth with examples:<br>
Acta Crystallogr. (2008), D64, 99-107.<br>
Acta Crystallogr. (2012), D68, 1541-1548.<br>
Acta Crystallogr. (2006), D62, 83-95.<br>
<br>
I hope this helps some people...<br>
<br>
Jon<br>
<br>
<pre class="moz-signature" cols="72">--
Jonathan P. Schuermann, Ph. D.
Beamline Scientist, NE-CAT
Argonne National Laboratory, 436E
9700 S. Cass Ave.
Argonne, IL 60439
Email: <a moz-do-not-send="true" class="moz-txt-link-abbreviated" href="mailto:schuerjp@anl.gov">schuerjp@anl.gov</a>
Tel: (630) 252-0682</pre>
<br>
<br>
<br>
<br>
On 05/30/2013 09:26 AM, Nathaniel Echols wrote:<br>
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cite="mid:CALeAa1N-vkxnfABKgY8efehr-siVz+MNYS=WmYjTYT1Xbwf0Ew@mail.gmail.com"
type="cite">
<pre wrap="">On Thu, May 30, 2013 at 7:09 AM, Heather Condurso <a moz-do-not-send="true" class="moz-txt-link-rfc2396E" href="mailto:condurso@bc.edu"><condurso@bc.edu></a> wrote:
</pre>
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<pre wrap="">I have a similar issue. My data is nearly perfectly twinned so Xtriage usually fails to find any twin law. However when I solve the structure in the lower symmetry space group with twinning my maps look beautiful. In the higher symmetry space group, only 3 of the 6 monomers fit into nice looking density. I simply use generate table 1 and uncheck the box so it won't regenerate R factors. The discrepancy between the R-factors is however much more significant than 2%. From refine I get 18/22 and from table1 28/31.
</pre>
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<pre wrap="">That's actually a little surprising that Xtriage wouldn't work - would
you be willing to send us the data? Keep in mind that if you have
perfect twinning and refine with the twin law, I think the maps will
look beautiful pretty much by definition, because of the model bias.
(Which doesn't mean that you did it wrong - the R-factors sound
realistic - you just need to be very careful and make lots of omit
maps.)
It helps to have actual examples when warning against the dangers of
twin refinement and model bias in general, so here's one (attached).
This map isn't beautiful, but it matches the model very closely,
including the complete lack of sidechains. Another Phenix user got
this result by accident with a mostly-if-not-entirely incorrect MR
solution - fortunately the combination of missing sidechain density
and relatively high R-free (~40%) prompted him to email us. I suspect
that if we searched the PDB we'd find an actual published example
(especially considering how many spurious ligands have been found
there).
(Sorry to sound like a broken record here, but this keeps coming up in
emails and workshops - perhaps we need better validation tools.)
-Nat
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