Dear all,
As I said in the latest topic, I could not model the third molecule.
But when I superpose the two trimmers found in P1 MR solution (link
below), I get the first two molecules perfect aligned and the third
molecule inverted! (It is also possible to see the 2-fold axis and the
third molecule lying on it!)
I tried to run a MR with a model with two alternative positions and
adjusted occupancy for the third molecule, but the Rfactor/free get
higher (> 40%) and the map becomes worse – even the good ones
(molecules 1 and 2) and for third molecule it remains bad (or worse).
A procedure that “solved” the problem (decreased the Rfactor/free and
gave good maps for third molecule) was the following: I integrated and
scaled the data in P21, then I ran the sfcheck and it showed a twinned
data (probably because of the (pseudo) higher symmetry present –
P21212). So, I detwinned the data (with detwinn) and run a MR with
detwinned data that gave a very good solution with tree molecules in
ASU (it have never happened before!). After the MR I refined this MR
solution against the original P21 data (without detwinn procedure)
with amplitude based twin refinement in Refmac5 and, finally, it gave
a good statistics (R factor / free about 0.19 / 0.22; FOM ~0.8) in the
first round of refinement. I think that procedure probably discard
reflections related to other positions making increasing the signal of
the most frequent position.
Link (.pdf): https://dl.dropbox.com/u/16221126/superposition.pdf
Is there some problem in procedure described? If so, does anybody have
a suggestion how can I model these disorder? Moreover, it seems to be
a long range disorder (multiples positions along the all lattice),
since even in P1 the maps for this third molecule are very bad.
Thank you for all the suggestions.
Cheers,
Andrey
2013/3/25 Eleanor Dodson <eleanor.dod...@york.ac.uk
<mailto:eleanor.dod...@york.ac.uk>>
First - I dont think you have a 3rd molecule where you have put it
- or at least not one with full occupancy. Those maps are a clear
indication that something is wrong. What is the Matthews
coefficient for the numbers in the asymmetric unit?
Presumably your processing gave you a lattice which fitted the
diffraction spots? ie you didnt miss a set of observations? You
should see that at the data processing stage, and the different
integration programs also try to report it. If there is
non-crystallographic translation that can confuse things a bit;
some classes of reflections might be systematically weak, but you
can find if there is such a phenomena by doing a patterson. Or run
ctruncate after merging the data - it checks this, and so does
Xtriage. All these options will also check for twinning. If there
is NCT then that could explain the high Rfactor.
Are the spots nicely shaped? There are some cases of sheared
crystals, which usually show up in distorted diffraction spots.
If this is so and you have integrated the data according to an
orthogonal lattice, there is nothing to stop you merging those
observations in a low symmetry. Pointless gives you good
statistics on the scoring for different symmetry operators.
You can either run MR again in that symmetry - check all SGS
consistent with the pointgroup, or try to work out how to position
your P22121 solution in the new SG. There may well be 2n+1 copies
of your molecule when you double the size of the asymmetric unit
- all hard to check without more information.
Good luck Eleanor
On 22 March 2013 17:54, Andrey Nascimento
<andreynascime...@gmail.com <mailto:andreynascime...@gmail.com>>
wrote:
Dear all,
I have tried the procedure recommended by Zbyszek, expanding
data from a higher symmetry and keeping the R-free set. But
the map for third molecule (new molecule placed) are still
very bad, even when a tried to reprocess data in P1 or P2 (P 1
21 1). The previous placed molecule (present in P2 21 21 ASU)
and its symmetry related on P21 shows a very good map, but the
third molecule are almost completely wrong (~50 residues in
470 are placed in quite good map) and map does not have
connectivity to build a new molecule (even in lower sigmas,
0.8-1.0). I have tried automatic model building (AutoBuild and
ARP/wARP) but they cannot build anything that make some sense
or build a random chains without any sense.
I do not have an extensive knowledge of crystallography, but I
have been thinking about some questions:
If the third molecule (the bad one) is lying on the 2-fold
symmetry axis on P 2 21 21, and since it does not have an
intrinsic 2-fold symmetry axis (like protein molecule), how
can I merge the structure factors (or intensities) related by
symmetry and expand to lower symmetry afterwards? In this case
the molecule lying on the 2-fold symmetry axis will have the
structure factors wrongly merged, since the molecule is not
symmetric, is it ok?
If the third molecule is lying on the 2-fold symmetry axis on
P 2 21 21, and only another two molecules can be related by
the crystallographic symmetry, is it a case of
pseudo-symmetry? But in this case, the third molecule is
disordered in the crystal packing (as Zbyszek said), and
probably have a long range disorder, because I cannot get a
good maps for this third molecule even in P1. (pseudo-symmetry
+ order/disorder????).
And a more philosophical question… what is the problem in
process data in a lower symmetry? Are there
mathematical/statistical problems related that can lead to
“false-good” data?
I put a new .pdf file (ccp4bb_maps_P21.pdf) with map figures
in this link:
https://dl.dropbox.com/u/16221126/ccp4bb_maps_P21.pdf
I am sorry for so many questions and thanks in advance.
Cheers,
Andrey
2013/3/20 Jrh <jrhelliw...@gmail.com
<mailto:jrhelliw...@gmail.com>>
Dear Zbyszek,
I am concerned that the unmerged data would be bypassed
and not preserved in your recommendation. I also find it
counter intuitive that the merged data would then be
unmerged into a lower symmetry and be better than the
unmerged data; there is I imagine some useful reference or
two you can direct me to that may well correct my lack of
understanding. Thirdly I think this a very likely useful
case to preserve the raw diffraction images.
All best wishes,
John
Prof John R Helliwell DSc
On 19 Mar 2013, at 14:37, Zbyszek Otwinowski
<zbys...@work.swmed.edu <mailto:zbys...@work.swmed.edu>>
wrote:
> It is a clear-cut case of crystal packing disorder. The
tell-tale sign is
> that data can be merged in the higher-symmetry lattice,
while the number
> of molecules in the asymmetric unit (3 in P21) is not
divisible by the
> higher symmetry factor (2, by going from P21 to P21212).
> From my experience, this is more likely a case of
order-disorder than
> merohedral twinning. The difference between these two is
that structure
> factors are added for the alternative conformations in
the case of
> order-disorder, while intensities (structure factors
squared) are added in
> the case of merohedral twinning.
>
> Now an important comment on how to proceed in the cases
where data can be
> merged in a higher symmetry, but the structure needs to
be solved in a
> lower symmetry due to a disorder.
>
> !Such data needs to be merged in the higher
symmetry,assigned R-free flag,
> and THEN expanded to the lower symmetry. Reprocessing
the data in a lower
> symmetry is an absolutely wrong procedure and it will
artificially reduce
> R-free, as the new R-free flags will not follow data
symmetry!
>
> Moreover, while this one is likely to be a case of
order-disorder, and
> these are infrequent, reprocessing the data in a lower
symmetry seems to
> be frequently abused, essentially in order to reduce
R-free. Generally,
> when data CAN be merged in a higher symmetry, the only
proper procedure in
> going to a lower-symmetry structure is by expanding
these higher-symmetry
> data to a lower symmetry, and not by rescaling and
merging the data in a
> lower symmetry.
>
> Zbyszek Otwinowski
>
>> Dear all,
>> We have solved the problem. Data processing in P1 looks
better (six
>> molecules in ASU), and Zanuda shows a P 1 21 1 symmetry
(three molecules
>> in
>> ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the
first round
>> of refinement (without put waters, ligands, etc.).
>>
>> Indeed, there were one more molecule in ASU, but the
over-merged data in
>> an orthorhombic lattice hid the correct solution.
>>
>> Thank you very much for all your suggestions, they were
very important to
>> solve this problem.
>>
>> Cheers,
>>
>> Andrey
>>
>> 2013/3/15 Andrey Nascimento <andreynascime...@gmail.com
<mailto:andreynascime...@gmail.com>>
>>
>>> *Dear all,*
>>>
>>> *I have collected a good quality dataset of a protein
with 64% of
>>> solvent
>>> in P 2 21 21 space group at 1.7A resolution with good
statistical
>>> parameters (values for last shell: Rmerge=0.202;
I/Isig.=4.4;
>>> Complet.=93%
>>> Redun.=2.4, the overall values are better than last
shell). The
>>> structure
>>> solution with molecular replacement goes well, the map
quality at the
>>> protein chain is very good, but in the final of
refinement, after
>>> addition
>>> of a lot of waters and other solvent molecules, TLS
refinement, etc. ...
>>> the Rfree is a quite high yet, considering this resolution
>>> (1.77A).(Rfree=
>>> 0.29966 and Rfactor= 0.25534). Moreover, I reprocess
the data in a lower
>>> symmetry space group (P21), but I got the same
problem, and I tried all
>>> possible space groups for P222, but with other screw
axis I can not even
>>> solve the structure.*
>>>
>>> *A strange thing in the structure are the large
solvent channels with a
>>> lot of electron density positive peaks!? I usually did
not see too many
>>> peaks in the solvent channel like this. This peaks are
the only reason
>>> for
>>> these high R's in refinement that I can find. But, why
are there too
>>> many
>>> peaks in the solvent channel???*
>>>
>>> *I put a .pdf file (ccp4bb_maps.pdf) with some more
information and map
>>> figures in this link:
https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf*
>>>
>>> *
>>> *
>>>
>>> *Do someone have an explanation or solution for this?*
>>>
>>> * *
>>>
>>> *Cheers,*
>>>
>>> *Andrey*
>>>
>>
>
>
> Zbyszek Otwinowski
> UT Southwestern Medical Center at Dallas
> 5323 Harry Hines Blvd.
> Dallas, TX 75390-8816
> Tel. 214-645-6385 <tel:214-645-6385>
> Fax. 214-645-6353 <tel:214-645-6353>
