Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Andrey Nascimento]

Dear Herman,
I would like to mention one more information, maybe I have forgotten. When
a process the data in P21212 and run the sfcheck it do not appear to have
twinning (even when a ran phenix Xtriage with older process data in
P21212). I will send direct to your e-mail the .pdf file with sfcheck
analysis to both space groups.
Thank you very much.
Andrey

2013/3/28 Andrey Nascimento 
>
>> Dear Appu,
>> I am sorry, I do not have the script file. I ran it basically from
>> default parameters in CCP4 GUI. I just ran the sfcheck for 'check
>> experimental data only' and I got the twin law and twin fraction from the
>> output (post script file). So, I put these information on the detwin and
>> ran it. I will send a .pdf file with the a print screen of ccp4 GUI.
>> I am not a experienced crystallographer, but I hope it helps you.
>> Good luck,
>> Andrey
>>
>>
>> 2013/3/28 Appu kumar 
>>
>>>
>>> Respected sir,
>>> I have same problem what you have. I am running
>>> the detwin on mtz file but it getting failed. Could you please tell me how
>>> you did this. If possible send me your script file. It will be a great help
>>> for me.
>>> Thank you in advance.
>>> Appu
>>>
>>> On 28 March 2013 05:10, Andrey Nascimento wrote:
>>>
 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 

> 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

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Andrey Nascimento]

Dear Appu,
I am sorry, I do not have the script file. I ran it basically from default
parameters in CCP4 GUI. I just ran the sfcheck for 'check experimental data
only' and I got the twin law and twin fraction from the output (post script
file). So, I put these information on the detwin and ran it. I will send a
.pdf file with the a print screen of ccp4 GUI.
I am not a experienced crystallographer, but I hope it helps you.
Good luck,
Andrey


2013/3/28 Appu kumar 

>
> Respected sir,
> I have same problem what you have. I am running
> the detwin on mtz file but it getting failed. Could you please tell me how
> you did this. If possible send me your script file. It will be a great help
> for me.
> Thank you in advance.
> Appu
>
> On 28 March 2013 05:10, Andrey Nascimento wrote:
>
>> 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 
>>
>>> 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 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 mole

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Herman . Schreuder]

Dear Andrey,

Due to our company policy, I cannot view the dropbox image, but the packing you 
describe is exactly what Edward Barry described and what I expected. Two 
molecules obey the 2-fold symmetry and one molecule is lying on a 2-fold with 
two possible superimposed orientations. The question is: are there frequent 
switches between both conformations such that X-rays diffracted from both 
conformations are interfering, or are there just larger chunks of crystal with 
either conformation A or B, behaving like separate crystals? In the first case, 
one has a crystal packing disorder and, as Eleanor was hinting at, some of the 
diffraction spots may look funny or smeared, in the second case one has 
twinning.

I do not know the exact method sfcheck uses, but normally to detect twinning, 
programs do not look at symmetry but look at intensity distributions. E.g. in 
case of twinning, intensities are averaged and less reflections will have 
extreme (very high or very low) values and reflections will have more average 
values. I would expect that if you expand an untwinned P21212 data set to P21 
and run the twin test, it still will come out untwinned. So if sfcheck claims 
the data is twinned, it most likely is and you have used the correct procedure. 
And in this case you should also process the data in the lower symmetry space 
group.

What wonders me a little is that MR did not give clear solutions in P21 with 
twinned data. P21 is low symmetry and I would have expected 2 clear solutions, 
each corresponding to one of the two twin possibilities. Also, if the detwin 
procedure does what I think it does, it should not perform well if the twin 
fraction is very close to 0.5 (perfect twinning). As has been pointed out by 
others, you will have to generate the freeR flags in P21212 and expand them to 
P21, otherwise your free reflections are linked to working reflections and you 
will get artificially low free Rfactors. Also, Rfactors and free Rfactors will 
generally be lower if you use twin refinement. No reflections are discarded 
during twin refinement, what happens is that both twin orientations are taken 
into account during refinement (and for the calculation of Rfactors!).

Congratulations, it looks like you have solved your problem!
Herman




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrey 
Nascimento
Sent: Thursday, March 28, 2013 12:41 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

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 
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-crystallo

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Frank von Delft]

Seems reasonable:  P21 with beta=90 is pretty common, and it often ends 
up twinned as well.


Most importantly, your R-factors support the hypothesis.

(Only keep in mind that R-factors are lower in twinned refinement - look 
on Garib's webpage for the presentation where he describes that -- 
sorry, I don't remember the exact slides.)


phx


On 27/03/2013 23:40, Andrey Nascimento wrote:


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 >


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
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
hav

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Andrey Nascimento]

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 

> 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 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 tw

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Eleanor Dodson]

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 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 
>
>> 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 
>> 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

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Andrey Nascimento]

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 

> 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 
> 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 
> >>
> >>> *Dear all,*

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Jrh]

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  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 
>> 
>>> *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
> Fax. 214-645-6353

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Herman . Schreuder]

Dear Edward,
I think you gave a very good summary of what might have happened in the 
crystals. If the two domains diffract like a single crystal and interfere, F's 
are added, if they diffract like two different crystals, I's are added. In 
fact, one should model the "special" protein molecule in two alternative 
orientations, just like one does with individual amino acids that have multiple 
conformations.  
Herman

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Edward A. 
Berry
Sent: Tuesday, March 19, 2013 7:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

Maybe this thread still needs some more pedagogy/explanation for those newbies 
and for biologist/ wanna-be crystallographers like me. My original reaction 
was- if the true space group is P21 you wouldn't want to expand from data 
reduced in higher symmetry, because you would be enforcing that higher symmetry.

But if it were simply a case of P21 symmetry, with three molecules in the AU, 
that happened to have a beta angle of 90, merging statistics would have 
prevented reducing the data in p222 in the first place.

Does order/disorder mean that the third molecule is actually present in two 
different orientations with equal occupancy, so that on the average it does 
obey the higher symmetry? Like our "heme on a special position" in the 
bacterioferritin paper?
And structure factors add because the two orientations are present in the same 
domain, whereas with twinning the two orientations are present in different 
domains that diffract like separate crystals, and the resulting intensities add 
on the "film"?


herman.schreu...@sanofi.com wrote:
> If it is crystal packing disorder (F's added instead of I's), the switches 
> between the alternative conformations need to be very frequent, to be within 
> coherent range, so I would asume that the alternative conformations will be 
> present in equal proportions. Still the alternatives need to be modeled 
> somehow and if this can be conveniently done in a lower symmetry spacegroup 
> this would not artificially lower the free R-factors. As Phoebe mentioned, 
> ignoring the higher symmetry relations and repicking the free Rflags at lower 
> symmetry would lead free reflections to be linked to the working set, leading 
> to too low Rfree values. However, with perfect packing disorder, no extra 
> information would be gained by reprocessing in lower symmetry (in contrast to 
> cases with pseudo symmetry).
>
> My 2 cents,
> Herman
>
> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
> Phoebe A. Rice
> Sent: Tuesday, March 19, 2013 4:49 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Strange density in solvent channel and high 
> Rfree
>
> oops, I should have expanded my comments to include the sort of funky lattice 
> order-disorder Zbyszek so cleverly diagnosed.  Scratch that "perfect 
> twinning" comment in my last message.
> 
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe 
> A. Rice [pr...@uchicago.edu]
> Sent: Tuesday, March 19, 2013 10:34 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Strange density in solvent channel and high 
> Rfree
>
> Hi Zbyszek,
>If the issue is perfect twinning, I agree - good point!
>But you don't want to confuse people who simply have nearly-but-not-quite 
> crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of 
> newbies read the BB).  We had a case of P31 that was so close to P61 we 
> actually solved the molecular replacement problem in P61, then expanded it 
> back and re-rigid-bodied it.  We've played similar games with translational 
> pseudo-symmetry (ignoring the weak spots at first).  In cases like that it is 
> important to properly reprocess the data in the lower symmetry space group 
> (or smaller unit cell) because there is real information in those small 
> differences.  However, the point about Rfree holds for twinning or rotational 
> pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry 
> operators, not re-picked in the lower symmetry space group.
> Phoebe
>
> ++
>
> Phoebe A. Rice
> Dept. of Biochemistry&  Molecular Biology The University of Chicago
> 773 834 1723; pr...@uchicago.edu
> http://bmb.bsd.uchicago.edu/Faculty_and_Research/
> http://www.rsc.org/shop/books/2008/9780854042722.asp
>
> ____________________
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek 
> Otwinowski [zbys...@work.swmed.edu]
> Sent: Tuesd

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Edward A. Berry]

Maybe this thread still needs some more pedagogy/explanation for those newbies and for biologist/ wanna-be 
crystallographers like me. My original reaction was- if the true space group is P21 you wouldn't want to expand from 
data reduced in higher symmetry, because you would be enforcing that higher symmetry.


But if it were simply a case of P21 symmetry, with three molecules in the AU, that happened to have a beta angle of 90, 
merging statistics would have prevented reducing the data in p222 in the first place.


Does order/disorder mean that the third molecule is actually present in two different orientations with equal occupancy, 
so that on the average it does obey the higher symmetry? Like our "heme on a special position" in the bacterioferritin 
paper?
And structure factors add because the two orientations are present in the same domain, whereas with twinning the two 
orientations are present in different domains that diffract like separate crystals, and the resulting intensities add on 
the "film"?



herman.schreu...@sanofi.com wrote:

If it is crystal packing disorder (F's added instead of I's), the switches 
between the alternative conformations need to be very frequent, to be within 
coherent range, so I would asume that the alternative conformations will be 
present in equal proportions. Still the alternatives need to be modeled somehow 
and if this can be conveniently done in a lower symmetry spacegroup this would 
not artificially lower the free R-factors. As Phoebe mentioned, ignoring the 
higher symmetry relations and repicking the free Rflags at lower symmetry would 
lead free reflections to be linked to the working set, leading to too low Rfree 
values. However, with perfect packing disorder, no extra information would be 
gained by reprocessing in lower symmetry (in contrast to cases with pseudo 
symmetry).

My 2 cents,
Herman

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phoebe A. 
Rice
Sent: Tuesday, March 19, 2013 4:49 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

oops, I should have expanded my comments to include the sort of funky lattice 
order-disorder Zbyszek so cleverly diagnosed.  Scratch that "perfect twinning" 
comment in my last message.

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice 
[pr...@uchicago.edu]
Sent: Tuesday, March 19, 2013 10:34 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

Hi Zbyszek,
   If the issue is perfect twinning, I agree - good point!
   But you don't want to confuse people who simply have nearly-but-not-quite 
crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of 
newbies read the BB).  We had a case of P31 that was so close to P61 we 
actually solved the molecular replacement problem in P61, then expanded it back 
and re-rigid-bodied it.  We've played similar games with translational 
pseudo-symmetry (ignoring the weak spots at first).  In cases like that it is 
important to properly reprocess the data in the lower symmetry space group (or 
smaller unit cell) because there is real information in those small 
differences.  However, the point about Rfree holds for twinning or rotational 
pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry 
operators, not re-picked in the lower symmetry space group.
Phoebe

++

Phoebe A. Rice
Dept. of Biochemistry&  Molecular Biology The University of Chicago
773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/
http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek 
Otwinowski [zbys...@work.swmed.edu]
Sent: Tuesday, March 19, 2013 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

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 

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Herman . Schreuder]

If it is crystal packing disorder (F's added instead of I's), the switches 
between the alternative conformations need to be very frequent, to be within 
coherent range, so I would asume that the alternative conformations will be 
present in equal proportions. Still the alternatives need to be modeled somehow 
and if this can be conveniently done in a lower symmetry spacegroup this would 
not artificially lower the free R-factors. As Phoebe mentioned, ignoring the 
higher symmetry relations and repicking the free Rflags at lower symmetry would 
lead free reflections to be linked to the working set, leading to too low Rfree 
values. However, with perfect packing disorder, no extra information would be 
gained by reprocessing in lower symmetry (in contrast to cases with pseudo 
symmetry).

My 2 cents,
Herman

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phoebe A. 
Rice
Sent: Tuesday, March 19, 2013 4:49 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

oops, I should have expanded my comments to include the sort of funky lattice 
order-disorder Zbyszek so cleverly diagnosed.  Scratch that "perfect twinning" 
comment in my last message.

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice 
[pr...@uchicago.edu]
Sent: Tuesday, March 19, 2013 10:34 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

Hi Zbyszek,
  If the issue is perfect twinning, I agree - good point!
  But you don't want to confuse people who simply have nearly-but-not-quite 
crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of 
newbies read the BB).  We had a case of P31 that was so close to P61 we 
actually solved the molecular replacement problem in P61, then expanded it back 
and re-rigid-bodied it.  We've played similar games with translational 
pseudo-symmetry (ignoring the weak spots at first).  In cases like that it is 
important to properly reprocess the data in the lower symmetry space group (or 
smaller unit cell) because there is real information in those small 
differences.  However, the point about Rfree holds for twinning or rotational 
pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry 
operators, not re-picked in the lower symmetry space group.
   Phoebe

++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology The University of Chicago
773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/
http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek 
Otwinowski [zbys...@work.swmed.edu]
Sent: Tuesday, March 19, 2013 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

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 

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Phoebe A. Rice]

oops, I should have expanded my comments to include the sort of funky lattice 
order-disorder Zbyszek so cleverly diagnosed.  Scratch that "perfect twinning" 
comment in my last message.

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice 
[pr...@uchicago.edu]
Sent: Tuesday, March 19, 2013 10:34 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

Hi Zbyszek,
  If the issue is perfect twinning, I agree - good point!
  But you don't want to confuse people who simply have nearly-but-not-quite 
crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of 
newbies read the BB).  We had a case of P31 that was so close to P61 we 
actually solved the molecular replacement problem in P61, then expanded it back 
and re-rigid-bodied it.  We've played similar games with translational 
pseudo-symmetry (ignoring the weak spots at first).  In cases like that it is 
important to properly reprocess the data in the lower symmetry space group (or 
smaller unit cell) because there is real information in those small 
differences.  However, the point about Rfree holds for twinning or rotational 
pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry 
operators, not re-picked in the lower symmetry space group.
   Phoebe

++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/
http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek 
Otwinowski [zbys...@work.swmed.edu]
Sent: Tuesday, March 19, 2013 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

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 
>
>> *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, a

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Jacob Keller]

Isn't lowering the symmetry equivalent to using multiple
models/conformations for one map? I remember seeing this done with the
infamous MSBA structure from a few years ago, so caveat emptor I guess. And
further, wouldn't using strict NCS make things equivalent to the
higher-symmetry space group? And then violating the NCS in places would
then just be equivalent to modelling multiple conformations, no?

JPK

On Tue, Mar 19, 2013 at 11:34 AM, Phoebe A. Rice  wrote:

> Hi Zbyszek,
>   If the issue is perfect twinning, I agree - good point!
>   But you don't want to confuse people who simply have
> nearly-but-not-quite crystallographic symmetry (OK, I'm being a bit
> pedagogical here, but a lot of newbies read the BB).  We had a case of P31
> that was so close to P61 we actually solved the molecular replacement
> problem in P61, then expanded it back and re-rigid-bodied it.  We've played
> similar games with translational pseudo-symmetry (ignoring the weak spots
> at first).  In cases like that it is important to properly reprocess the
> data in the lower symmetry space group (or smaller unit cell) because there
> is real information in those small differences.  However, the point about
> Rfree holds for twinning or rotational pseudo-symmetry: the Rfree flags
> should be expanded by the xtal symmetry operators, not re-picked in the
> lower symmetry space group.
>Phoebe
>
> ++
>
> Phoebe A. Rice
> Dept. of Biochemistry & Molecular Biology
> The University of Chicago
> 773 834 1723; pr...@uchicago.edu
> http://bmb.bsd.uchicago.edu/Faculty_and_Research/
> http://www.rsc.org/shop/books/2008/9780854042722.asp
>
> 
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek
> Otwinowski [zbys...@work.swmed.edu]
> Sent: Tuesday, March 19, 2013 9:37 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree
>
> 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 
> >
> >> *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 q

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Phoebe A. Rice]

Hi Zbyszek,
  If the issue is perfect twinning, I agree - good point!
  But you don't want to confuse people who simply have nearly-but-not-quite 
crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of 
newbies read the BB).  We had a case of P31 that was so close to P61 we 
actually solved the molecular replacement problem in P61, then expanded it back 
and re-rigid-bodied it.  We've played similar games with translational 
pseudo-symmetry (ignoring the weak spots at first).  In cases like that it is 
important to properly reprocess the data in the lower symmetry space group (or 
smaller unit cell) because there is real information in those small 
differences.  However, the point about Rfree holds for twinning or rotational 
pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry 
operators, not re-picked in the lower symmetry space group.
   Phoebe

++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/
http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek 
Otwinowski [zbys...@work.swmed.edu]
Sent: Tuesday, March 19, 2013 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

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 
>
>> *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

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Zbyszek Otwinowski]

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 
>
>> *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
Fax. 214-645-6353

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Andrey Nascimento]

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 

> *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*
>

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Kendall Nettles]

I second Phoebe's suggestion. Looks like another molecule to me. If you are 
doing molecular replacement you may need to get creative about trimming. When 
we had a case like that it wasn't until the third person worked on it that we 
go a solution because he trimmed the model differently than the first two who 
tried.

Kendall

On Mar 15, 2013, at 3:29 PM, "Phoebe A. Rice" 
mailto:pr...@uchicago.edu>> wrote:


What happens if you solvent-flatten/flip/massage that map, but tell the 
software the solvent content much lower than what you think it is now?  Maybe 
you'll find another copy of the molecule?




From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] 
on behalf of Andrey Nascimento 
[andreynascime...@gmail.com<mailto:andreynascime...@gmail.com>]
Sent: Friday, March 15, 2013 1:39 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] Strange density in solvent channel and high Rfree

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

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Edward A. Berry]

Or give it to arp/warp, either with the current model to improve or with phases
from the current model to build new model?

Phoebe A. Rice wrote:

What happens if you solvent-flatten/flip/massage that map, but tell the 
software the solvent content much lower than
what you think it is now? Maybe you'll find another copy of the molecule?


*From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Andrey 
Nascimento [andreynascime...@gmail.com]
*Sent:* Friday, March 15, 2013 1:39 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] Strange density in solvent channel and high Rfree

*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*

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Parthasarathy Sampathkumar]

Hi Andrey,

I am taking a risky guess:

>From your slide #2, it looks like a termini (unless this correspond to the
beginning or end of disordered a loop) of the protein chain(s) is (are)
extending toward(s) the solvent channel. Are the density features shown in
slides #3,4, and 5 extend from this terminal residue?!! If this is true,
did you missed to model any uncleaved-tag residues?!! I would also look at
2Fo-Fc at 1.0 sigma.

Hope this helps,
Partha


On Fri, Mar 15, 2013 at 2:39 PM, Andrey Nascimento <
andreynascime...@gmail.com> wrote:

> *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*
>

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Phoebe A. Rice]

What happens if you solvent-flatten/flip/massage that map, but tell the 
software the solvent content much lower than what you think it is now?  Maybe 
you'll find another copy of the molecule?




From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Andrey 
Nascimento [andreynascime...@gmail.com]
Sent: Friday, March 15, 2013 1:39 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Strange density in solvent channel and high Rfree

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

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Francis E Reyes]

Wow, Usually on the default settings of adxv, I can see spots at this I/sig! 

I suspect your data goes higher than 1.77. 

Include more of the high resolution data.  You very likely don't have the 
correct space group. 

F

On Mar 15, 2013, at 11:39 AM, Andrey Nascimento  
wrote:

>  I/Isig.=4.4



-
Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Ed Pozharski]

Check for translational NCS

And you are way too conservative with resolution.  Even those holding
onto the Rmerge-dictated past would probably acquiesce to lower I/sig
cutoff.  If you are using aimless, follow its recommendations based on
CC1/2, it's good for you.

Cheers,

Ed.

On Fri, 2013-03-15 at 15:39 -0300, Andrey Nascimento wrote:
> 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
> 

-- 
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

[ccp4bb] Strange density in solvent channel and high Rfree

[Andrey Nascimento]

*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*