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*

Re: [ccp4bb] Strange density

[Dale Tronrud]

   No such luck!  If one calculated the Root Mean Square Deviation from
the Mean then F(000) makes no difference, but everyone I know calculates
the Deviation from 0.0.  I guess that makes it an "rms" and not an
"rmsd".

   We can use maps calculated w/o the F(000) because we are generally
more interested in the shape of the density than its height.  We use
the shape to come up with interpretations, which is the hard part.
The height can give us a clue about the occupancy an atom would have
if we refined one there - It is a shortcut to avoid building and
refining models that are destined to be nonsense.  If the molecule
you are building will refine to an occupancy of 0.1 you could spend
you time better by doing something else.

Dale Tronrud

On 11/28/12 15:13, Lijun Liu wrote:
> F000 contributes to the whole map as a level (F000/V).  If calculated
> with a only difference of with or w/o F000, should the sigma levels of
> the two maps be the same?   That is why we could rely on maps for
> modeling that are calculated w/o F000 item.   Lijun
> 
> 
> On Nov 28, 2012, at 2:30 PM, Pavel Afonine wrote:
> 
>> For map in e-/A^3 units to make sense one needs to obtain F000, which
>> may be more tricky than one may think. Interesting, how Coot does this
>> given just a set of Fourier map coefficients?
>>
>> Pavel
>>
>> On Wed, Nov 28, 2012 at 12:21 PM, Greg Costakes > <mailto:gcost...@purdue.edu>> wrote:
>>
>> You stated that the map is set to 3 sigma, but what is the
>> e-/A^3?  In Coot I often find that my fo-fc map needs to be maxed
>> out (max sigma) in order to get to an acceptable e-/A^3. It is
>> possible that your fo-fc map at 3 sigma has an e-/A^3 of 0.04 or
>> something low like that.
>>
>> 
>> ---
>> Greg Costakes
>> PhD Candidate
>> Department of Structural Biology
>> Purdue University
>> Hockmeyer Hall, Room 320
>> 240 S. Martin Jischke Drive, West Lafayette, IN 47907
>>
>> 
>> 
>>
>>
>> --------
>> *From: *"Jon Read" > <mailto:jon.r...@astrazeneca.com>>
>> *To: *CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
>> *Sent: *Wednesday, November 28, 2012 10:48:04 AM
>> *Subject: *[ccp4bb] Strange density
>>
>>
>>
>> Anyone see anything like this before? The data is 1.7Angstrom data
>> with good statistics. The picture shows the solid FoFc density
>> contoured at 3  Sigma in light brown and -3 Sigma in purple. The
>> density is odd as it appears to be bound to a peptide carbonyl
>> with no other obvious interactions with the protein. There is a
>> characteristic tail at one end.
>>
>>  
>>
>>  
>>  
>>
>>
>>
>> 
>>
>> AstraZeneca UK Limited is a company incorporated in England and
>> Wales with registered number: 03674842 and a registered office at
>> 2 Kingdom Street, London, W2 6BD.
>>
>>
>> *Confidentiality Notice: *This message is private and may contain
>> confidential, proprietary and legally privileged information. If
>> you have received this message in error, please notify us and
>> remove it from your system and note that you must not copy,
>> distribute or take any action in reliance on it. Any unauthorised
>> use or disclosure of the contents of this message is not permitted
>> and may be unlawful.
>>
>>
>> *Disclaimer:* Email messages may be subject to delays,
>> interception, non-delivery and unauthorised alterations.
>> Therefore, information expressed in this message is not given or
>> endorsed by AstraZeneca UK Limited unless otherwise notified by an
>> authorised representative independent of this message. No
>> contractual relationship is created by this message by any person
>> unless specifically indicated by agreement in writing other than
>> email.
>>
>>
>> *Monitoring: *AstraZeneca UK Limited may monitor email traffic
>> data and content for the purposes of the prevention and detection
>> of crime, ensuring the security of our computer systems and
>> checking compliance with our Code of Conduct and policies.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
> 

Re: [ccp4bb] Strange density

[Lijun Liu]

F000 contributes to the whole map as a level (F000/V).  If calculated with a 
only difference of with or w/o F000, should the sigma levels of the two maps be 
the same?   That is why we could rely on maps for modeling that are calculated 
w/o F000 item.   Lijun


On Nov 28, 2012, at 2:30 PM, Pavel Afonine wrote:

> For map in e-/A^3 units to make sense one needs to obtain F000, which may be 
> more tricky than one may think. Interesting, how Coot does this given just a 
> set of Fourier map coefficients?
> 
> Pavel
> 
> On Wed, Nov 28, 2012 at 12:21 PM, Greg Costakes  wrote:
> You stated that the map is set to 3 sigma, but what is the e-/A^3?  In Coot I 
> often find that my fo-fc map needs to be maxed out (max sigma) in order to 
> get to an acceptable e-/A^3. It is possible that your fo-fc map at 3 sigma 
> has an e-/A^3 of 0.04 or something low like that. 
> 
> ---
> Greg Costakes
> PhD Candidate
> Department of Structural Biology
> Purdue University
> Hockmeyer Hall, Room 320
> 240 S. Martin Jischke Drive, West Lafayette, IN 47907
> 
> 
> 
> 
> From: "Jon Read" 
> To: CCP4BB@JISCMAIL.AC.UK
> Sent: Wednesday, November 28, 2012 10:48:04 AM
> Subject: [ccp4bb] Strange density
> 
> 
> 
> Anyone see anything like this before? The data is 1.7Angstrom data with good 
> statistics. The picture shows the solid FoFc density contoured at 3  Sigma in 
> light brown and -3 Sigma in purple. The density is odd as it appears to be 
> bound to a peptide carbonyl with no other obvious interactions with the 
> protein. There is a characteristic tail at one end.
> 
>  
> 
> 
>  
>  
> 
> 
> 
> AstraZeneca UK Limited is a company incorporated in England and Wales with 
> registered number: 03674842 and a registered office at 2 Kingdom Street, 
> London, W2 6BD.
> 
> 
> Confidentiality Notice: This message is private and may contain confidential, 
> proprietary and legally privileged information. If you have received this 
> message in error, please notify us and remove it from your system and note 
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> Monitoring: AstraZeneca UK Limited may monitor email traffic data and content 
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> 
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> 
> 
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> 
> 
> 
> 
> 
> 
> 
> 
> 

Re: [ccp4bb] Strange density

[Dale Tronrud]

ity
> > Hockmeyer Hall, Room 320
> > 240 S. Martin Jischke Drive, West Lafayette, IN 47907
> >
> >
> 
> 
> >
> >
> >
> ------------
> > *From: *"Jon Read"  <mailto:jon.r...@astrazeneca.com>
> > <mailto:jon.r...@astrazeneca.com
> <mailto:jon.r...@astrazeneca.com>>>
> > *To: *CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
> <mailto:CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>>
> > *Sent: *Wednesday, November 28, 2012 10:48:04 AM
> > *Subject: *[ccp4bb] Strange density
> >
> >
> > Anyone see anything like this before? The data is 1.7Angstrom data
> > with good statistics. The picture shows the solid FoFc density
> > contoured at 3  Sigma in light brown and -3 Sigma in purple. The
> > density is odd as it appears to be bound to a peptide carbonyl
> with
> > no other obvious interactions with the protein. There is a
> > characteristic tail at one end.
> >
> >
> >
> >
> >
> >
> >
> >
> 
> >
> > AstraZeneca UK Limited is a company incorporated in England and
> > Wales with registered number: 03674842 and a registered office
> at 2
> > Kingdom Street, London, W2 6BD.
> >
> > *Confidentiality Notice: *This message is private and may contain
> > confidential, proprietary and legally privileged information.
> If you
> > have received this message in error, please notify us and
> remove it
> > from your system and note that you must not copy, distribute
> or take
> > any action in reliance on it. Any unauthorised use or
> disclosure of
> > the contents of this message is not permitted and may be unlawful.
> >
> > *Disclaimer:* Email messages may be subject to delays,
> interception,
> > non-delivery and unauthorised alterations. Therefore, information
> > expressed in this message is not given or endorsed by
> AstraZeneca UK
> > Limited unless otherwise notified by an authorised representative
> > independent of this message. No contractual relationship is
> created
> > by this message by any person unless specifically indicated by
> > agreement in writing other than email.
> >
> > *Monitoring: *AstraZeneca UK Limited may monitor email traffic
> data
> > and content for the purposes of the prevention and detection of
> > crime, ensuring the security of our computer systems and checking
> > compliance with our Code of Conduct and policies.
> >
> >
> 
> 

Re: [ccp4bb] Strange density

[Pavel Afonine]

If a map was computed from map coefficients not having F000 included I
would not say it has *electron* per Angstrom cube (e/A**3) units, but it's
just a unit cell volume scaled map in some units.

Whether such a map more useful compared to sigma scaled one is a separate
question. In fact, it is, as you and Ed pointed out.

I was just trying to say that if one did not include F000 one should not
interpret map values too literally (thinking of them as electrons, etc).
The second point was that F000 is typically estimated (not experimentally
measured), and any estimation errors will be in turn reflected in e/A**3
map values, which adds one more reason of being cautious when interpreting
map values.

Pavel

On Wed, Nov 28, 2012 at 2:13 PM, Dale Tronrud wrote:

>
>Actually one can make a lot of sense about e/A^3 in the absence of
> F(000).  You actually think of the density as difference from the
> average and not an absolute measurement.
>
>For an Fo-Fc style map the F(000) term is simply the difference
> between the number of missing electrons in the model and the number
> of extra electrons.  Since we are probably missing all data of
> resolution lower than about 20 A because of the beamstop the model
> defects are only counted if they are within about 20 A or so of
> the point you are looking at.  In the latter stages of refinement,
> when one is trying to identify strange density, the rest of the
> model should be pretty good and the expected mean value of the
> difference map very near zero.  Of course your model is missing
> atoms for the blob itself so the difference density will tend to
> sink, resulting in somewhat lower peaks and negative density around
> the edges but this effect is usually not huge.
>
>On the other hand, contouring based on rmsd (i.e. sigma ack!)
> causes huge differences depending on the other things that are going
> on in your map.  The rmsd of your first difference map can be many
> times larger than it is in your last.  The density for a missing
> water molecule contoured at 3 rmsd in the first map will look very
> different than the same water molecule contoured at 3 rmsd in the
> last map.  That water molecule contoured at, say, 0.18 e/A^3 would
> look pretty much the same.
>
>In the first difference map that water molecule will be surrounded
> by a huge number of other features when you contour at 0.18 e/A^3
> and by very few in the last map, but isn't that as it should be?
> The map is supposed to be flatter at the end.
>
> Dale Tronrud
>
> On 11/28/12 12:30, Pavel Afonine wrote:
> > For map in e-/A^3 units to make sense one needs to obtain F000, which
> > may be more tricky than one may think. Interesting, how Coot does this
> > given just a set of Fourier map coefficients?
> >
> > Pavel
> >
> > On Wed, Nov 28, 2012 at 12:21 PM, Greg Costakes  > <mailto:gcost...@purdue.edu>> wrote:
> >
> > You stated that the map is set to 3 sigma, but what is the e-/A^3?
> > In Coot I often find that my fo-fc map needs to be maxed out (max
> > sigma) in order to get to an acceptable e-/A^3. It is possible that
> > your fo-fc map at 3 sigma has an e-/A^3 of 0.04 or something low
> > like that.
> >
> >
> ---
> > Greg Costakes
> > PhD Candidate
> > Department of Structural Biology
> > Purdue University
> > Hockmeyer Hall, Room 320
> > 240 S. Martin Jischke Drive, West Lafayette, IN 47907
> >
> >
> 
> >
> >
> >
> ------------
> > *From: *"Jon Read"  > <mailto:jon.r...@astrazeneca.com>>
> > *To: *CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
> > *Sent: *Wednesday, November 28, 2012 10:48:04 AM
> > *Subject: *[ccp4bb] Strange density
> >
> >
> > Anyone see anything like this before? The data is 1.7Angstrom data
> > with good statistics. The picture shows the solid FoFc density
> > contoured at 3  Sigma in light brown and -3 Sigma in purple. The
> > density is odd as it appears to be bound to a peptide carbonyl with
> > no other obvious interactions with the protein. There is a
> > characteristic tail at one end.
> >
> >
> >
> >
> >
> >
> >
> >
> 
> >
> > AstraZeneca UK Limited is a company incorporated in England and
> > Wales with regis

Re: [ccp4bb] Strange density

[Dale Tronrud]

   Actually one can make a lot of sense about e/A^3 in the absence of
F(000).  You actually think of the density as difference from the
average and not an absolute measurement.

   For an Fo-Fc style map the F(000) term is simply the difference
between the number of missing electrons in the model and the number
of extra electrons.  Since we are probably missing all data of
resolution lower than about 20 A because of the beamstop the model
defects are only counted if they are within about 20 A or so of
the point you are looking at.  In the latter stages of refinement,
when one is trying to identify strange density, the rest of the
model should be pretty good and the expected mean value of the
difference map very near zero.  Of course your model is missing
atoms for the blob itself so the difference density will tend to
sink, resulting in somewhat lower peaks and negative density around
the edges but this effect is usually not huge.

   On the other hand, contouring based on rmsd (i.e. sigma ack!)
causes huge differences depending on the other things that are going
on in your map.  The rmsd of your first difference map can be many
times larger than it is in your last.  The density for a missing
water molecule contoured at 3 rmsd in the first map will look very
different than the same water molecule contoured at 3 rmsd in the
last map.  That water molecule contoured at, say, 0.18 e/A^3 would
look pretty much the same.

   In the first difference map that water molecule will be surrounded
by a huge number of other features when you contour at 0.18 e/A^3
and by very few in the last map, but isn't that as it should be?
The map is supposed to be flatter at the end.

Dale Tronrud

On 11/28/12 12:30, Pavel Afonine wrote:
> For map in e-/A^3 units to make sense one needs to obtain F000, which
> may be more tricky than one may think. Interesting, how Coot does this
> given just a set of Fourier map coefficients?
> 
> Pavel
> 
> On Wed, Nov 28, 2012 at 12:21 PM, Greg Costakes  <mailto:gcost...@purdue.edu>> wrote:
> 
> You stated that the map is set to 3 sigma, but what is the e-/A^3? 
> In Coot I often find that my fo-fc map needs to be maxed out (max
> sigma) in order to get to an acceptable e-/A^3. It is possible that
> your fo-fc map at 3 sigma has an e-/A^3 of 0.04 or something low
> like that.
> 
> 
> ---
> Greg Costakes
> PhD Candidate
> Department of Structural Biology
> Purdue University
> Hockmeyer Hall, Room 320
> 240 S. Martin Jischke Drive, West Lafayette, IN 47907
> 
> 
> 
> 
> 
> 
> *From: *"Jon Read"  <mailto:jon.r...@astrazeneca.com>>
> *To: *CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
> *Sent: *Wednesday, November 28, 2012 10:48:04 AM
> *Subject: *[ccp4bb] Strange density
> 
> 
> Anyone see anything like this before? The data is 1.7Angstrom data
> with good statistics. The picture shows the solid FoFc density
> contoured at 3  Sigma in light brown and -3 Sigma in purple. The
> density is odd as it appears to be bound to a peptide carbonyl with
> no other obvious interactions with the protein. There is a
> characteristic tail at one end.
> 
>  
> 
>  
> 
>  
> 
> 
> 
> AstraZeneca UK Limited is a company incorporated in England and
> Wales with registered number: 03674842 and a registered office at 2
> Kingdom Street, London, W2 6BD.
> 
> *Confidentiality Notice: *This message is private and may contain
> confidential, proprietary and legally privileged information. If you
> have received this message in error, please notify us and remove it
> from your system and note that you must not copy, distribute or take
> any action in reliance on it. Any unauthorised use or disclosure of
> the contents of this message is not permitted and may be unlawful.
> 
> *Disclaimer:* Email messages may be subject to delays, interception,
> non-delivery and unauthorised alterations. Therefore, information
> expressed in this message is not given or endorsed by AstraZeneca UK
> Limited unless otherwise notified by an authorised representative
> independent of this message. No contractual relationship is created
> by this message by any person unless specifically indicated by
> agreement in writing other than email.
> 
> *Monitoring: *AstraZeneca UK Limited may monitor email traffic data
> and content for the purposes of the prevention and detection of
> crime, ensuring the security of our computer systems and checking
> compliance with our Code of Conduct and policies.
> 
> 

Re: [ccp4bb] Strange density

[Edward A. Berry]

Without F000 we cannot get absolute density level vs zero, but still
I think electron density in units of e-/a^3 above the mean is much more
meaningful than sigma above the mean. I think the last one of these 
mystery density questions generated a lot of discussion until someone 
pointed out that there was no 2Fo-Fc density for the feature.


eab

Pavel Afonine wrote:

For map in e-/A^3 units to make sense one needs to obtain F000, which
may be more tricky than one may think. Interesting, how Coot does this
given just a set of Fourier map coefficients?

Pavel

On Wed, Nov 28, 2012 at 12:21 PM, Greg Costakes mailto:gcost...@purdue.edu>> wrote:

You stated that the map is set to 3 sigma, but what is the e-/A^3?
In Coot I often find that my fo-fc map needs to be maxed out (max
sigma) in order to get to an acceptable e-/A^3. It is possible that
your fo-fc map at 3 sigma has an e-/A^3 of 0.04 or something low
like that.


---
Greg Costakes
PhD Candidate
Department of Structural Biology
Purdue University
Hockmeyer Hall, Room 320
240 S. Martin Jischke Drive, West Lafayette, IN 47907






*From: *"Jon Read" mailto:jon.r...@astrazeneca.com>>
*To: *CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
*Sent: *Wednesday, November 28, 2012 10:48:04 AM
    *Subject: *[ccp4bb] Strange density


Anyone see anything like this before? The data is 1.7Angstrom data
with good statistics. The picture shows the solid FoFc density
contoured at 3  Sigma in light brown and -3 Sigma in purple. The
density is odd as it appears to be bound to a peptide carbonyl with
no other obvious interactions with the protein. There is a
characteristic tail at one end.



AstraZeneca UK Limited is a company incorporated in England and
Wales with registered number: 03674842 and a registered office at 2
Kingdom Street, London, W2 6BD.

*Confidentiality Notice: *This message is private and may contain
confidential, proprietary and legally privileged information. If you
have received this message in error, please notify us and remove it
from your system and note that you must not copy, distribute or take
any action in reliance on it. Any unauthorised use or disclosure of
the contents of this message is not permitted and may be unlawful.

*Disclaimer:* Email messages may be subject to delays, interception,
non-delivery and unauthorised alterations. Therefore, information
expressed in this message is not given or endorsed by AstraZeneca UK
Limited unless otherwise notified by an authorised representative
independent of this message. No contractual relationship is created
by this message by any person unless specifically indicated by
agreement in writing other than email.

*Monitoring: *AstraZeneca UK Limited may monitor email traffic data
and content for the purposes of the prevention and detection of
crime, ensuring the security of our computer systems and checking
compliance with our Code of Conduct and policies.

Re: [ccp4bb] Strange density

[Pavel Afonine]

For map in e-/A^3 units to make sense one needs to obtain F000, which may
be more tricky than one may think. Interesting, how Coot does this given
just a set of Fourier map coefficients?

Pavel

On Wed, Nov 28, 2012 at 12:21 PM, Greg Costakes  wrote:

> You stated that the map is set to 3 sigma, but what is the e-/A^3?  In
> Coot I often find that my fo-fc map needs to be maxed out (max sigma) in
> order to get to an acceptable e-/A^3. It is possible that your fo-fc map at
> 3 sigma has an e-/A^3 of 0.04 or something low like that.
>
>
> ---
> Greg Costakes
> PhD Candidate
> Department of Structural Biology
> Purdue University
> Hockmeyer Hall, Room 320
> 240 S. Martin Jischke Drive, West Lafayette, IN 47907
>
>
> 
>
>
> --
> *From: *"Jon Read" 
> *To: *CCP4BB@JISCMAIL.AC.UK
> *Sent: *Wednesday, November 28, 2012 10:48:04 AM
> *Subject: *[ccp4bb] Strange density
>
>
> Anyone see anything like this before? The data is 1.7Angstrom data with
> good statistics. The picture shows the solid FoFc density contoured at 3
> Sigma in light brown and -3 Sigma in purple. The density is odd as it
> appears to be bound to a peptide carbonyl with no other obvious
> interactions with the protein. There is a characteristic tail at one end.
>
>
>
>
>
>
>
>  --
>
> AstraZeneca UK Limited is a company incorporated in England and Wales with
> registered number: 03674842 and a registered office at 2 Kingdom Street,
> London, W2 6BD.
>
> *Confidentiality Notice: *This message is private and may contain
> confidential, proprietary and legally privileged information. If you have
> received this message in error, please notify us and remove it from your
> system and note that you must not copy, distribute or take any action in
> reliance on it. Any unauthorised use or disclosure of the contents of this
> message is not permitted and may be unlawful.
>
> *Disclaimer:* Email messages may be subject to delays, interception,
> non-delivery and unauthorised alterations. Therefore, information expressed
> in this message is not given or endorsed by AstraZeneca UK Limited unless
> otherwise notified by an authorised representative independent of this
> message. No contractual relationship is created by this message by any
> person unless specifically indicated by agreement in writing other than
> email.
>
> *Monitoring: *AstraZeneca UK Limited may monitor email traffic data and
> content for the purposes of the prevention and detection of crime, ensuring
> the security of our computer systems and checking compliance with our Code
> of Conduct and policies.
>
>

Re: [ccp4bb] Strange density

[Dale Tronrud]

Hi,

   These sorts of questions are always difficult, particularly in the
absence of any information about the protein or the contents of the
mother liquor.  If the carbonyl you are talking about is the little
magenta dot visible through the hole in your blob, this could be a
metal atom with some long chelating molecule around the equator. In
the extreme it could be some sort of porphyrin, although the density
would be very poor if it was.

Dale Tronrud

On 11/28/2012 7:48 AM, Read, Jon wrote:

Anyone see anything like this before? The data is 1.7Angstrom data with good 
statistics. The picture shows the solid FoFc density contoured at 3  Sigma in 
light brown and -3 Sigma in purple. The density is odd as it appears to be 
bound to a peptide carbonyl with no other obvious interactions with the 
protein. There is a characteristic tail at one end.



AstraZeneca UK Limited is a company incorporated in England and Wales with 
registered number: 03674842 and a registered office at 2 Kingdom Street, 
London, W2 6BD.

*Confidentiality Notice: *This message is private and may contain confidential, 
proprietary and legally privileged information. If you have received this 
message in error, please notify us and remove it from your system and note that 
you must not copy, distribute or take any action in reliance on it. Any 
unauthorised use or disclosure of the contents of this message is not permitted 
and may be unlawful.

*Disclaimer:* Email messages may be subject to delays, interception, 
non-delivery and unauthorised alterations. Therefore, information expressed in 
this message is not given or endorsed by AstraZeneca UK Limited unless 
otherwise notified by an authorised representative independent of this message. 
No contractual relationship is created by this message by any person unless 
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Re: [ccp4bb] Strange density

[VAN RAAIJ , MARK JOHAN]

could it be a PEG molecule?

Quoting "Read, Jon":


Anyone see anything like this before? The data is 1.7Angstrom data with
good statistics. The picture shows the solid FoFc density contoured at 3
Sigma in light brown and -3 Sigma in purple. The density is odd as it
appears to be bound to a peptide carbonyl with no other obvious
interactions with the protein. There is a characteristic tail at one
end.










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Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
28049 Madrid
tel. 91 585 4616
email: mjvanra...@cnb.csic.es

Re: [ccp4bb] Strange Density

[Yuri Pompeu]

Grinter,

I would be very careful when comparing atomic distances (or even considering 
them) using a 3.1 A data set. Take a look at what error estimates are for this 
resolution, given your R values ;).
Also, I second what has been said, that you should build a model that makes 
"physical" sense, and plus, you have a good idea of what your protein  is like 
from your higher resolution holo-structure.
HTH

Re: [ccp4bb] Strange Density

[Jacob Keller]

How about chloride? I know, there are negative electrostatics, but I think
such is seen sometimes for halides. Or, is it possible that there is a side
chain flipped?

Jacob

On Tue, May 15, 2012 at 9:51 AM, RHYS GRINTER <
r.grinte...@research.gla.ac.uk> wrote:

> Dear Community,
>
> As I'm a relatively new to protein crystallography this might turn out to
> be an obvious question, however.
>
> I'm working on the structure of a enzyme requiring Ca2+ for activity and
> with calcium coordinated in the active site by Asp and 2x backbone carbonyl
> groups, in a crystal structure with Ca in the crystallisation conditions (
> http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_TD_15A.jpg).
> When Ca is omitted from the crystallizing conditions and a divalent
> chelator (EGTA) is added the crystals are of significantly lower resolution
> (3.13A). Refinement of this data reveals density for a molecule coordinated
> by the Ca coordinating Asp and backbone, however this density is
> significantly further away (3.4-3.8A) too far away for water or a strongly
> coordinated divalent cation(
> http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_EGTA_315.jpg). The
> density is also much weaker than for Ca in the previous model disappearing
> at 3.5 sigma.
>
> The crystallisation conditions for the Ca free condition is:
>
> 0.1M Tris/Bicine buffer [pH 8.5]
> 8% PEG 8000
> 30% Ethylene Glycol
> 1mM EGTA
>
> The protein was purified by nickel affinity/SEC and dialysed into:
> 20mM NaCl
> 20mM Tris [pH 8.0]
>
>
> A colleague suggested that sulphate or phosphate could fit at these
> distances, but these ions have not been added at any stage of the
> crystallisation process.
>
>
> Could anyone give me some insight into what this density might represent?
>
> Thanks in advance,
>
> Rhys Grinter
> PhD Candidate
> University of Glasgow
>



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] Strange Density

[Sanishvili, Ruslan]

Hi Rhis,
It may have been suggested already but still...
X-ray fluorescence spectra can often tell you what elements you may be dealing 
with. Spectra won't tell anything about binding specifics, but any extra bit of 
information could help. Any descent synchrotron MX beamline, equipped for 
MAD/SAD phasing, should be able to help. Of course, spectroscopy beamlines 
would be even better.
Cheers,
N.

Ruslan Sanishvili (Nukri), Ph.D.

GM/CA-CAT
Biosciences Division, ANL
9700 S. Cass Ave.
Argonne, IL 60439

Tel: (630)252-0665
Fax: (630)252-0667
rsanishv...@anl.gov

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of RHYS 
GRINTER
Sent: Tuesday, May 15, 2012 9:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Strange Density

Dear Community,

As I'm a relatively new to protein crystallography this might turn out to be an 
obvious question, however.

I'm working on the structure of a enzyme requiring Ca2+ for activity and with 
calcium coordinated in the active site by Asp and 2x backbone carbonyl groups, 
in a crystal structure with Ca in the crystallisation conditions 
(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_TD_15A.jpg). 
When Ca is omitted from the crystallizing conditions and a divalent chelator 
(EGTA) is added the crystals are of significantly lower resolution (3.13A). 
Refinement of this data reveals density for a molecule coordinated by the Ca 
coordinating Asp and backbone, however this density is significantly further 
away (3.4-3.8A) too far away for water or a strongly coordinated divalent 
cation(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_EGTA_315.jpg). The 
density is also much weaker than for Ca in the previous model disappearing at 
3.5 sigma.

The crystallisation conditions for the Ca free condition is:

0.1M Tris/Bicine buffer [pH 8.5]
8% PEG 8000
30% Ethylene Glycol
1mM EGTA

The protein was purified by nickel affinity/SEC and dialysed into: 
20mM NaCl 
20mM Tris [pH 8.0]


A colleague suggested that sulphate or phosphate could fit at these distances, 
but these ions have not been added at any stage of the crystallisation process. 


Could anyone give me some insight into what this density might represent?

Thanks in advance,

Rhys Grinter
PhD Candidate
University of Glasgow

Re: [ccp4bb] Strange Density

[Dale Tronrud]

   Your holo structure has a Ca++ and three water molecules that have not been
built into your low resolution apo map.  These atoms are not expected to be
resolved at 3 A resolution, so I would expect them to appear as a large, 
misshapened,
blob.  Your screenshot only shows one contour level.  It is quite possible that
the highest density value is not at the center of the blob.

   You might have a lower occupancy Ca++ atom at the site and the image is 
confused
by the low resolution.  Remember, even if the concentration of Ca++ is lower in
this mother liquor any Ca++ that binds will bind exactly as it does in the
fully occupied case.  A weakly binding Ca++ site will not bind before the 
strongly
binding site.

   I would first look to see what your holo map looks like when it's resolution 
is
truncated to 3 A.  This will give you a sense of what a Ca++ binding in this 
site
would look like.  You could try refining a model with the Ca++ and water 
molecules,
with lower occupancy, and see what the residual difference map looks like.  You 
will,
of course, have to have strong restraints on the geometry to hold this model 
together
at 3 A resolution, but fortunately you have a higher resolution model to base 
these
restraints on.

   The PDB file is a statement of your belief of what is in the crystal.  Don't 
waste
your time refining models that don't make chemical sense.  An ion floating in 
space
with no ligands is not a reasonable model so even if it "fits" the density it 
can't
be correct.

   There a multiple ways of justifying the model of a crystal and others on the 
list
will likely have different ideas for the criteria that should be used.  My 
belief is
that you know the holo model and the most likely outcome of your Ca++ extraction
experiment (in a Bayesian prior sense) is a lower occupancy binding of the Ca++ 
and
its water molecules.  If you build and refine that model and the difference map 
is
acceptable you can say that this model is consistent with your experiment.  If 
there
is residual density then you can conclude that something is replacing the Ca++,
but untangling superimposed, partial occupancy, models at 3.1 A resolution is
extremely difficult.  I think all you will be able to say is that "something
replaces the Ca++ but it cannot be identified".

   Not everything can be identified in a 3 A map.  Not everything can be 
identified
in a 1 A map.  Your job is to say "these parts I understand and these parts I 
don't".

Dale Tronrud

On 05/15/12 07:51, RHYS GRINTER wrote:
> Dear Community,
> 
> As I'm a relatively new to protein crystallography this might turn out to be 
> an obvious question, however.
> 
> I'm working on the structure of a enzyme requiring Ca2+ for activity and with 
> calcium coordinated in the active site by Asp and 2x backbone carbonyl 
> groups, in a crystal structure with Ca in the crystallisation conditions 
> (http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_TD_15A.jpg). 
> When Ca is omitted from the crystallizing conditions and a divalent chelator 
> (EGTA) is added the crystals are of significantly lower resolution (3.13A). 
> Refinement of this data reveals density for a molecule coordinated by the Ca 
> coordinating Asp and backbone, however this density is significantly further 
> away (3.4-3.8A) too far away for water or a strongly coordinated divalent 
> cation(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_EGTA_315.jpg). 
> The density is also much weaker than for Ca in the previous model 
> disappearing at 3.5 sigma.
> 
> The crystallisation conditions for the Ca free condition is:
> 
> 0.1M Tris/Bicine buffer [pH 8.5]
> 8% PEG 8000
> 30% Ethylene Glycol
> 1mM EGTA
> 
> The protein was purified by nickel affinity/SEC and dialysed into: 
> 20mM NaCl 
> 20mM Tris [pH 8.0]
> 
> 
> A colleague suggested that sulphate or phosphate could fit at these 
> distances, but these ions have not been added at any stage of the 
> crystallisation process. 
> 
> 
> Could anyone give me some insight into what this density might represent?
> 
> Thanks in advance,
> 
> Rhys Grinter
> PhD Candidate
> University of Glasgow

Re: [ccp4bb] Strange Density

[Ed Pozharski]

On Tue, 2012-05-15 at 15:51 +0100, RHYS GRINTER wrote:
> A colleague suggested that sulphate or phosphate could fit at these
> distances, but these ions have not been added at any stage of the
> crystallisation process. 
> 

I vaguely remember a report about 2-3 years ago at the ACA meeting of
phosphate contamination of PEGs from certain manufacturers.  But why
would phosphate (negatively charged) bind at the site that seems to
attract calcium (positively charged)?  Potassium?

Check if you have a peak at that position in anomalous difference map.
Maybe ICP-MS can give you an answer.

-- 
I don't know why the sacrifice thing didn't work.  
Science behind it seemed so solid.
Julian, King of Lemurs

Re: [ccp4bb] Strange Density

[Kelly Daughtry]

Try refining with: Na, Ca or Water at that position and compare the
resulting maps. That should provide you with the information you need.

It could be a weakly bound sodium, or calcium ion. It could be that
calcium was not fully removed by EGTA treatment.

Kelly
***
Kelly Daughtry, Ph.D.
Post-Doctoral Fellow, Raetz Lab
Biochemistry Department
Duke University
Alex H. Sands, Jr. Building
303 Research Drive
RM 250
Durham, NC 27710
P: 919-684-5178
***


On Tue, May 15, 2012 at 10:51 AM, RHYS GRINTER
 wrote:
> Dear Community,
>
> As I'm a relatively new to protein crystallography this might turn out to be 
> an obvious question, however.
>
> I'm working on the structure of a enzyme requiring Ca2+ for activity and with 
> calcium coordinated in the active site by Asp and 2x backbone carbonyl 
> groups, in a crystal structure with Ca in the crystallisation conditions 
> (http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_TD_15A.jpg).
> When Ca is omitted from the crystallizing conditions and a divalent chelator 
> (EGTA) is added the crystals are of significantly lower resolution (3.13A). 
> Refinement of this data reveals density for a molecule coordinated by the Ca 
> coordinating Asp and backbone, however this density is significantly further 
> away (3.4-3.8A) too far away for water or a strongly coordinated divalent 
> cation(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_EGTA_315.jpg). 
> The density is also much weaker than for Ca in the previous model 
> disappearing at 3.5 sigma.
>
> The crystallisation conditions for the Ca free condition is:
>
> 0.1M Tris/Bicine buffer [pH 8.5]
> 8% PEG 8000
> 30% Ethylene Glycol
> 1mM EGTA
>
> The protein was purified by nickel affinity/SEC and dialysed into:
> 20mM NaCl
> 20mM Tris [pH 8.0]
>
>
> A colleague suggested that sulphate or phosphate could fit at these 
> distances, but these ions have not been added at any stage of the 
> crystallisation process.
>
>
> Could anyone give me some insight into what this density might represent?
>
> Thanks in advance,
>
> Rhys Grinter
> PhD Candidate
> University of Glasgow

[ccp4bb] Strange Density

[RHYS GRINTER]

Dear Community,

As I'm a relatively new to protein crystallography this might turn out to be an 
obvious question, however.

I'm working on the structure of a enzyme requiring Ca2+ for activity and with 
calcium coordinated in the active site by Asp and 2x backbone carbonyl groups, 
in a crystal structure with Ca in the crystallisation conditions 
(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_TD_15A.jpg). 
When Ca is omitted from the crystallizing conditions and a divalent chelator 
(EGTA) is added the crystals are of significantly lower resolution (3.13A). 
Refinement of this data reveals density for a molecule coordinated by the Ca 
coordinating Asp and backbone, however this density is significantly further 
away (3.4-3.8A) too far away for water or a strongly coordinated divalent 
cation(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_EGTA_315.jpg). The 
density is also much weaker than for Ca in the previous model disappearing at 
3.5 sigma.

The crystallisation conditions for the Ca free condition is:

0.1M Tris/Bicine buffer [pH 8.5]
8% PEG 8000
30% Ethylene Glycol
1mM EGTA

The protein was purified by nickel affinity/SEC and dialysed into: 
20mM NaCl 
20mM Tris [pH 8.0]


A colleague suggested that sulphate or phosphate could fit at these distances, 
but these ions have not been added at any stage of the crystallisation process. 


Could anyone give me some insight into what this density might represent?

Thanks in advance,

Rhys Grinter
PhD Candidate
University of Glasgow

Re: [ccp4bb] Strange density in Arg

[Eleanor Dodson]

On 07/04/2011 04:24 PM, ruheng wrote:
> 
> Dear all,
> 
> Recently we are working on an archaebacteria protein which was expressed and 
> purified from E.coli by conventional procedures. After we solved the 
> structure, we found that there is an extra density in one of the argninine as 
> shown in the attached picture. It seems that the density is larger than a 
> methyl group and the atoms in the density are not on one plane. So we are 
> curious about whether the density is a kind of posttranslational modification 
> of arginine residues in the protein, if it is, what kind of modification 
> could it be? And most important, what is the biological significance of this 
> kind modification? Any suggestions and dicussions are appreciated!
> 
> Best,
> Heng
>   
We saw something like this and modelled it as glycerol. But I dont know
how one knows if one is correct!!
 Eleanor

Re: [ccp4bb] Strange density in arginine

[Shiva Bhowmik]

Hi Ruheng,

Curious to know what was in the crystallization cocktail, cryoprotectant?
Did you collect the dataset at a synchrotron?

Shiva

2011/7/4 ruheng 

>  Dear all,
>
> Recently we are working on an archaebacteria protein which was expressed
> and purified from *E.coli *by conventional procedures. After we solved the
> structure, we found that there is an extra density in one of the argninine
> as shown in the attached picture. It seems that the density is larger than a
> methyl group and the atoms in the density are not on one plane. So we are
> curious about whether the density is a kind of posttranslational
> modification of arginine residues in the protein, if it is, what kind of
> modification could it be? And most important, what is the biological
> significance of this kind modification? Any suggestions and dicussions are
> appreciated!
>
> Best,
> Heng
>
> 
>  Strange density in arginine
> 
>   
> 查看幻灯片
> 下载全部
> 添加更多照片
> 此相册包含 1 张照片，并且将在 2011/10/02 之前在 SkyDrive 上提供。
>通过 Hotmail 共享您自己的幻灯片 
>

Re: [ccp4bb] Strange density in Arg

[Miri Hirshberg]

Mon. July 4th 2011
EBI

Good evening,

firstly, it could be something that came form the buffer/crystallisation 
soup etc. What about phosphate?


Secondly, if you think it is a "natural" post-translation modification of 
your specific protein this is gene/source depended. Check the uniprot 
entry of your protein, UNP does lists the known modification. Is there 
a mass-spec analysis?


Miri

On Mon, 4 Jul 2011, Artem Evdokimov wrote:


Before ruling this a covalent modification, did you check if a sulfate or some 
other tetrahedral ion (or
a disordered acetate) fit the bill with respect to distances to the neighboring 
atoms?
 
Artem

2011/7/4 ruheng 
  Dear all,
   
  Recently we are working on an archaebacteria protein which was expressed 
and purified from
  E.coli by conventional procedures. After we solved the structure, we 
found that there is an
  extra density in one of the argninine as shown in the attached picture. 
It seems that the
  density is larger than a methyl group and the atoms in the density are 
not on one plane. So
  we are curious about whether the density is a kind of posttranslational 
modification of
  arginine residues in the protein, if it is, what kind of modification 
could it be? And most
  important, what is the biological significance of this kind modification? 
Any suggestions and
  dicussions are appreciated!
   
  Best,
  Heng






Mon. July 4th, 2011
EBI

Re: [ccp4bb] Strange density in Arg

[Artem Evdokimov]

Before ruling this a covalent modification, did you check if a sulfate or
some other tetrahedral ion (or a disordered acetate) fit the bill with
respect to distances to the neighboring atoms?

Artem

2011/7/4 ruheng 

>  Dear all,
>
> Recently we are working on an archaebacteria protein which was expressed
> and purified from E.coli by conventional procedures. After we solved the
> structure, we found that there is an extra density in one of the argninine
> as shown in the attached picture. It seems that the density is larger than a
> methyl group and the atoms in the density are not on one plane. So we are
> curious about whether the density is a kind of posttranslational
> modification of arginine residues in the protein, if it is, what kind of
> modification could it be? And most important, what is the biological
> significance of this kind modification? Any suggestions and dicussions are
> appreciated!
>
> Best,
> Heng
>

Re: [ccp4bb] Strange density in arginine

[Roger Rowlett]

Looks like an anion, possibly sulfate or phosphate. What's in the
crystallization mix?

Roger Rowlett
On Jul 4, 2011 11:33 AM, "ruheng"  wrote:
>
> Dear all,
>
> Recently we are working on an archaebacteria protein which was expressed
and purified from E.coli by conventional procedures. After we solved the
structure, we found that there is an extra density in one of the argninine
as shown in the attached picture. It seems that the density is larger than a
methyl group and the atoms in the density are not on one plane. So we are
curious about whether the density is a kind of posttranslational
modification of arginine residues in the protein, if it is, what kind of
modification could it be? And most important, what is the biological
significance of this kind modification? Any suggestions and dicussions are
appreciated!
>
> Best,
> Heng
> Strange density in arginine
>
http://skydrive.live.com/redir.aspx?cid=2f5a8d98562e91e4&page=browse&resid=2F5A8D98562E91E4!198&type=5&Bpub=SDX.Photos&Bsrc=Photomail&authkey=S1J8JneduIs%24

Re: [ccp4bb] Strange density in arginine

[Vellieux Frederic]

The distance to your modelled water seems to be in agreement with an
ionic interaction. What are the components of all buffers in the
crystallisation components ?

Fred.

ruheng wrote:
> Dear all,
>
> Recently we are working on an archaebacteria protein which was
> expressed and purified from /E.coli /by conventional procedures. After
> we solved the structure, we found that there is an extra density in
> one of the argninine as shown in the attached picture. It seems that
> the density is larger than a methyl group and the atoms in the density
> are not on one plane. So we are curious about whether the density is a
> kind of posttranslational modification of arginine residues in the
> protein, if it is, what kind of modification could it be? And most
> important, what is the biological significance of this kind
> modification? Any suggestions and dicussions are appreciated!
>
> Best,
> Heng
> 
>   
> Strange density in arginine
> 
>
> 查看幻灯片
> 
>   下载全部
> 
>
> 添加更多照片
> 
>
>
> 此相册包含 1 张照片，并且将在 2011/10/02 之前在 SkyDrive 上提供。
>
> 通过 Hotmail 共享您自己的幻灯片 

Re: [ccp4bb] strange density

[Harry Powell]

Hi folks

Whoops, brain not engaged before hitting "send". 

Lewis acid/bases are to do with electron-pair donor/acceptance, Brønsted 
acid/bases with protons. So my second two paragraphs define things the wrong 
way round.

I need a coffee...

On 25 Feb 2011, at 10:49, Harry Powell wrote:

> Hi
> 
> Before people get carried away, it may be useful to mention that we are 
> discussing Lewis acids & bases here, not Brønsted (or Brønsted-Lowry) acids 
> and bases. 
> 
> Simply put, Brønsted acids are electron pair acceptors whereas Lewis acids 
> are proton donors. Some (not all)  Lewis acids are also Brønsted acids.
> 
> On the other hand, Lewis bases (proton acceptor) are all Brønsted bases 
> (electron pair donor).
> 
> 
> On 24 Feb 2011, at 23:28, Michael Thompson wrote:
> 
>> Jacob,
>> 
>> Roger is correct, this concept does refer to the Pearson HSAB theory. To 
>> summarize: This theory is applicable outside of inorganic chemistry as well, 
>> but it is extremely useful for explaining coordination chemistry of 
>> metal-ligand complexes. The theory states that "hard" acids interact with 
>> "hard" bases and "soft" acids interact with "soft" bases to form a bond that 
>> is covalent-like in nature. "Hardness" vs. "softness" is based on the 
>> energetic properties of the HOMO and LUMO of the acid and base. Generally 
>> hard acids/bases have small atomic/ionic radii, low polarizability, and high 
>> electronegativity whereas "soft" acids/bases tend to have larger radii, high 
>> polarizability, and low electronegativity. 
>> 
>> Hard bases are things like carboxylates, whereas soft bases are things like 
>> thiolates. Ligands with nitrogen (imidazole) are often in the middle 
>> somewhere.
>> 
>> Hard acids are ions like Na+, K+, Mg2+, etc., while soft acids are metals 
>> like mercury, silver, etc. Again, many biologically relevant things lie in 
>> the middle of the spectrum somewhere (Fe, Co, Zn).
>> 
>> It is possible to calculate the "chemical hardness" of a species, but that's 
>> where my knowledge stops.
>> 
>> Hope this is helpful,
>> 
>> Mike
>> 
>> 
>> 
>> 
>> - Original Message -
>> From: "Jacob Keller" 
>> To: CCP4BB@JISCMAIL.AC.UK
>> Sent: Thursday, February 24, 2011 10:39:09 AM GMT -08:00 US/Canada Pacific
>> Subject: Re: [ccp4bb] strange density
>> 
>> I have heard "hard" and "soft" many times now about O's and N's--to
>> what property of those ligands does this metaphor refer?
>> 
>> JPK
>> 
>> On Thu, Feb 24, 2011 at 12:47 PM, Jeffrey D Brodin  wrote:
>>> Alex,
>>> 
>>> I modeled in the bis-tris with the tertiary amine and and his imidazole
>>> coordinating axially and the four oxygens coordinating in the equatorial
>>> plane. However, it's hard for me to tell from your images if there are two
>>> His coordinating? Either way, that crescent shape could easily be explained
>>> by a bis-tris molecule, you'll just have to figure out how exactly to model
>>> it in. It's also possible that the metal is a Mg, but as people have already
>>> mentioned, nitrogens probably wouldn't coordinate very tightly to a hard
>>> metal. Lastly, I'm also not sure off the top of my head how tightly bis-tris
>>> binds metals, but it should be an easy number to look up. Hope this helps,
>>> 
>>> Jeff
>>> On Feb 24, 2011, at 9:02 AM, Alex Singer wrote:
>>> 
>>>> Hi -- thank you for all your help.  The majority opinion seems to be a
>>>> metal for the sphere (Ni from the Ni-affinity column, which (Joe
>>>> Patel, correct) was used during purification, but Zn and Fe were also
>>>> mentioned), and either water molecules, bis-tris or some other small
>>>> molecule forming the crescent.  Just looking at the density, the
>>>> occupancy would seem to be quite high, so I'm surprised that a Ni ion
>>>> (or a contaminating metal ion) could have gone through the
>>>> purification and still remained at high enough concentration to be
>>>> clearly visible in the crystals.  However, I'll still try this but
>>>> first some points of clarification and questions which you can either
>>>> email me seperately or post to the the group.
>>>> 
>>>> a.  it was collected at beamline 19-BM at Argonne, so radiation damage
>>>> is an issue.  Thierry Fishmann -- 

Re: [ccp4bb] strange density

[Harry Powell]

Hi

Before people get carried away, it may be useful to mention that we are 
discussing Lewis acids & bases here, not Brønsted (or Brønsted-Lowry) acids and 
bases. 

Simply put, Brønsted acids are electron pair acceptors whereas Lewis acids are 
proton donors. Some (not all)  Lewis acids are also Brønsted acids.

On the other hand, Lewis bases (proton acceptor) are all Brønsted bases 
(electron pair donor).


On 24 Feb 2011, at 23:28, Michael Thompson wrote:

> Jacob,
> 
> Roger is correct, this concept does refer to the Pearson HSAB theory. To 
> summarize: This theory is applicable outside of inorganic chemistry as well, 
> but it is extremely useful for explaining coordination chemistry of 
> metal-ligand complexes. The theory states that "hard" acids interact with 
> "hard" bases and "soft" acids interact with "soft" bases to form a bond that 
> is covalent-like in nature. "Hardness" vs. "softness" is based on the 
> energetic properties of the HOMO and LUMO of the acid and base. Generally 
> hard acids/bases have small atomic/ionic radii, low polarizability, and high 
> electronegativity whereas "soft" acids/bases tend to have larger radii, high 
> polarizability, and low electronegativity. 
> 
> Hard bases are things like carboxylates, whereas soft bases are things like 
> thiolates. Ligands with nitrogen (imidazole) are often in the middle 
> somewhere.
> 
> Hard acids are ions like Na+, K+, Mg2+, etc., while soft acids are metals 
> like mercury, silver, etc. Again, many biologically relevant things lie in 
> the middle of the spectrum somewhere (Fe, Co, Zn).
> 
> It is possible to calculate the "chemical hardness" of a species, but that's 
> where my knowledge stops.
> 
> Hope this is helpful,
> 
> Mike
> 
> 
> 
> 
> - Original Message -
> From: "Jacob Keller" 
> To: CCP4BB@JISCMAIL.AC.UK
> Sent: Thursday, February 24, 2011 10:39:09 AM GMT -08:00 US/Canada Pacific
> Subject: Re: [ccp4bb] strange density
> 
> I have heard "hard" and "soft" many times now about O's and N's--to
> what property of those ligands does this metaphor refer?
> 
> JPK
> 
> On Thu, Feb 24, 2011 at 12:47 PM, Jeffrey D Brodin  wrote:
>> Alex,
>> 
>> I modeled in the bis-tris with the tertiary amine and and his imidazole
>> coordinating axially and the four oxygens coordinating in the equatorial
>> plane. However, it's hard for me to tell from your images if there are two
>> His coordinating? Either way, that crescent shape could easily be explained
>> by a bis-tris molecule, you'll just have to figure out how exactly to model
>> it in. It's also possible that the metal is a Mg, but as people have already
>> mentioned, nitrogens probably wouldn't coordinate very tightly to a hard
>> metal. Lastly, I'm also not sure off the top of my head how tightly bis-tris
>> binds metals, but it should be an easy number to look up. Hope this helps,
>> 
>> Jeff
>> On Feb 24, 2011, at 9:02 AM, Alex Singer wrote:
>> 
>>> Hi -- thank you for all your help.  The majority opinion seems to be a
>>> metal for the sphere (Ni from the Ni-affinity column, which (Joe
>>> Patel, correct) was used during purification, but Zn and Fe were also
>>> mentioned), and either water molecules, bis-tris or some other small
>>> molecule forming the crescent.  Just looking at the density, the
>>> occupancy would seem to be quite high, so I'm surprised that a Ni ion
>>> (or a contaminating metal ion) could have gone through the
>>> purification and still remained at high enough concentration to be
>>> clearly visible in the crystals.  However, I'll still try this but
>>> first some points of clarification and questions which you can either
>>> email me seperately or post to the the group.
>>> 
>>> a.  it was collected at beamline 19-BM at Argonne, so radiation damage
>>> is an issue.  Thierry Fishmann -- for the gln residue, there was
>>> difference density for the gamma carbon after the first conformation
>>> was modeled in, thus the addition of the second conformation, which I
>>> agree is suspect.  What does the radiation damage do chemically and
>>> would that make the gamma carbon more mobile?
>>> 
>>> b.  Jeffrey D Brodin -- how did you model in the bis-tris?  Looking at
>>> the bis-tris molecule from Hic-up, was the N at the centre of the
>>> crescent and the O6 and O8 at the edges?
>>> 
>>> c.  JR Helliwell -- there are 4 molecules in the AU, but two H138&#

Re: [ccp4bb] strange density

[Michael Thompson]

Jacob,

Roger is correct, this concept does refer to the Pearson HSAB theory. To 
summarize: This theory is applicable outside of inorganic chemistry as well, 
but it is extremely useful for explaining coordination chemistry of 
metal-ligand complexes. The theory states that "hard" acids interact with 
"hard" bases and "soft" acids interact with "soft" bases to form a bond that is 
covalent-like in nature. "Hardness" vs. "softness" is based on the energetic 
properties of the HOMO and LUMO of the acid and base. Generally hard 
acids/bases have small atomic/ionic radii, low polarizability, and high 
electronegativity whereas "soft" acids/bases tend to have larger radii, high 
polarizability, and low electronegativity. 

Hard bases are things like carboxylates, whereas soft bases are things like 
thiolates. Ligands with nitrogen (imidazole) are often in the middle somewhere.

Hard acids are ions like Na+, K+, Mg2+, etc., while soft acids are metals like 
mercury, silver, etc. Again, many biologically relevant things lie in the 
middle of the spectrum somewhere (Fe, Co, Zn).

It is possible to calculate the "chemical hardness" of a species, but that's 
where my knowledge stops.

Hope this is helpful,

Mike




- Original Message -
From: "Jacob Keller" 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Thursday, February 24, 2011 10:39:09 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] strange density

I have heard "hard" and "soft" many times now about O's and N's--to
what property of those ligands does this metaphor refer?

JPK

On Thu, Feb 24, 2011 at 12:47 PM, Jeffrey D Brodin  wrote:
> Alex,
>
> I modeled in the bis-tris with the tertiary amine and and his imidazole
> coordinating axially and the four oxygens coordinating in the equatorial
> plane. However, it's hard for me to tell from your images if there are two
> His coordinating? Either way, that crescent shape could easily be explained
> by a bis-tris molecule, you'll just have to figure out how exactly to model
> it in. It's also possible that the metal is a Mg, but as people have already
> mentioned, nitrogens probably wouldn't coordinate very tightly to a hard
> metal. Lastly, I'm also not sure off the top of my head how tightly bis-tris
> binds metals, but it should be an easy number to look up. Hope this helps,
>
> Jeff
> On Feb 24, 2011, at 9:02 AM, Alex Singer wrote:
>
>> Hi -- thank you for all your help.  The majority opinion seems to be a
>> metal for the sphere (Ni from the Ni-affinity column, which (Joe
>> Patel, correct) was used during purification, but Zn and Fe were also
>> mentioned), and either water molecules, bis-tris or some other small
>> molecule forming the crescent.  Just looking at the density, the
>> occupancy would seem to be quite high, so I'm surprised that a Ni ion
>> (or a contaminating metal ion) could have gone through the
>> purification and still remained at high enough concentration to be
>> clearly visible in the crystals.  However, I'll still try this but
>> first some points of clarification and questions which you can either
>> email me seperately or post to the the group.
>>
>> a.  it was collected at beamline 19-BM at Argonne, so radiation damage
>> is an issue.  Thierry Fishmann -- for the gln residue, there was
>> difference density for the gamma carbon after the first conformation
>> was modeled in, thus the addition of the second conformation, which I
>> agree is suspect.  What does the radiation damage do chemically and
>> would that make the gamma carbon more mobile?
>>
>> b.  Jeffrey D Brodin -- how did you model in the bis-tris?  Looking at
>> the bis-tris molecule from Hic-up, was the N at the centre of the
>> crescent and the O6 and O8 at the edges?
>>
>> c.  JR Helliwell -- there are 4 molecules in the AU, but two H138's
>> are pointing into the solvent.  Thus the molar ratio of protein
>> molecules to "thing 1" is 4:1.  Also looking at the images, I see no
>> ice rings -- the images look pretty good.  Can you tell me more about
>> the series termination effects?
>>
>> Again thank you for your help and I'll let the group know how it worked
>> out.
>>
>> Alex
>>
>> --
>> Dr. Alex Singer
>> C.H. Best Institute
>> 112 College St. Room 70
>> University of Toronto
>> Toronto, Canada, M5G 1L6
>> 416-978-4033
>



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

-- 
Michael C. Thompson

Graduate Student

Biochemistry & Molecular Biology Division

Department of Chemistry & Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu

Re: [ccp4bb] strange density

[Roger Rowlett]

If I recall correctly this refers to the Pearson (?) hard/soft acid base
theory. It's been a long time since inorganic chemistry.

Cheers, Roger Rowlett
On Feb 24, 2011 1:39 PM, "Jacob Keller" 
wrote:
> I have heard "hard" and "soft" many times now about O's and N's--to
> what property of those ligands does this metaphor refer?
>
> JPK
>
> On Thu, Feb 24, 2011 at 12:47 PM, Jeffrey D Brodin 
wrote:
>> Alex,
>>
>> I modeled in the bis-tris with the tertiary amine and and his imidazole
>> coordinating axially and the four oxygens coordinating in the equatorial
>> plane. However, it's hard for me to tell from your images if there are
two
>> His coordinating? Either way, that crescent shape could easily be
explained
>> by a bis-tris molecule, you'll just have to figure out how exactly to
model
>> it in. It's also possible that the metal is a Mg, but as people have
already
>> mentioned, nitrogens probably wouldn't coordinate very tightly to a hard
>> metal. Lastly, I'm also not sure off the top of my head how tightly
bis-tris
>> binds metals, but it should be an easy number to look up. Hope this
helps,
>>
>> Jeff
>> On Feb 24, 2011, at 9:02 AM, Alex Singer wrote:
>>
>>> Hi -- thank you for all your help.  The majority opinion seems to be a
>>> metal for the sphere (Ni from the Ni-affinity column, which (Joe
>>> Patel, correct) was used during purification, but Zn and Fe were also
>>> mentioned), and either water molecules, bis-tris or some other small
>>> molecule forming the crescent.  Just looking at the density, the
>>> occupancy would seem to be quite high, so I'm surprised that a Ni ion
>>> (or a contaminating metal ion) could have gone through the
>>> purification and still remained at high enough concentration to be
>>> clearly visible in the crystals.  However, I'll still try this but
>>> first some points of clarification and questions which you can either
>>> email me seperately or post to the the group.
>>>
>>> a.  it was collected at beamline 19-BM at Argonne, so radiation damage
>>> is an issue.  Thierry Fishmann -- for the gln residue, there was
>>> difference density for the gamma carbon after the first conformation
>>> was modeled in, thus the addition of the second conformation, which I
>>> agree is suspect.  What does the radiation damage do chemically and
>>> would that make the gamma carbon more mobile?
>>>
>>> b.  Jeffrey D Brodin -- how did you model in the bis-tris?  Looking at
>>> the bis-tris molecule from Hic-up, was the N at the centre of the
>>> crescent and the O6 and O8 at the edges?
>>>
>>> c.  JR Helliwell -- there are 4 molecules in the AU, but two H138's
>>> are pointing into the solvent.  Thus the molar ratio of protein
>>> molecules to "thing 1" is 4:1.  Also looking at the images, I see no
>>> ice rings -- the images look pretty good.  Can you tell me more about
>>> the series termination effects?
>>>
>>> Again thank you for your help and I'll let the group know how it worked
>>> out.
>>>
>>> Alex
>>>
>>> --
>>> Dr. Alex Singer
>>> C.H. Best Institute
>>> 112 College St. Room 70
>>> University of Toronto
>>> Toronto, Canada, M5G 1L6
>>> 416-978-4033
>>
>
>
>
> --
> ***
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> cel: 773.608.9185
> email: j-kell...@northwestern.edu
> ***

Re: [ccp4bb] strange density

[Jacob Keller]

I have heard "hard" and "soft" many times now about O's and N's--to
what property of those ligands does this metaphor refer?

JPK

On Thu, Feb 24, 2011 at 12:47 PM, Jeffrey D Brodin  wrote:
> Alex,
>
> I modeled in the bis-tris with the tertiary amine and and his imidazole
> coordinating axially and the four oxygens coordinating in the equatorial
> plane. However, it's hard for me to tell from your images if there are two
> His coordinating? Either way, that crescent shape could easily be explained
> by a bis-tris molecule, you'll just have to figure out how exactly to model
> it in. It's also possible that the metal is a Mg, but as people have already
> mentioned, nitrogens probably wouldn't coordinate very tightly to a hard
> metal. Lastly, I'm also not sure off the top of my head how tightly bis-tris
> binds metals, but it should be an easy number to look up. Hope this helps,
>
> Jeff
> On Feb 24, 2011, at 9:02 AM, Alex Singer wrote:
>
>> Hi -- thank you for all your help.  The majority opinion seems to be a
>> metal for the sphere (Ni from the Ni-affinity column, which (Joe
>> Patel, correct) was used during purification, but Zn and Fe were also
>> mentioned), and either water molecules, bis-tris or some other small
>> molecule forming the crescent.  Just looking at the density, the
>> occupancy would seem to be quite high, so I'm surprised that a Ni ion
>> (or a contaminating metal ion) could have gone through the
>> purification and still remained at high enough concentration to be
>> clearly visible in the crystals.  However, I'll still try this but
>> first some points of clarification and questions which you can either
>> email me seperately or post to the the group.
>>
>> a.  it was collected at beamline 19-BM at Argonne, so radiation damage
>> is an issue.  Thierry Fishmann -- for the gln residue, there was
>> difference density for the gamma carbon after the first conformation
>> was modeled in, thus the addition of the second conformation, which I
>> agree is suspect.  What does the radiation damage do chemically and
>> would that make the gamma carbon more mobile?
>>
>> b.  Jeffrey D Brodin -- how did you model in the bis-tris?  Looking at
>> the bis-tris molecule from Hic-up, was the N at the centre of the
>> crescent and the O6 and O8 at the edges?
>>
>> c.  JR Helliwell -- there are 4 molecules in the AU, but two H138's
>> are pointing into the solvent.  Thus the molar ratio of protein
>> molecules to "thing 1" is 4:1.  Also looking at the images, I see no
>> ice rings -- the images look pretty good.  Can you tell me more about
>> the series termination effects?
>>
>> Again thank you for your help and I'll let the group know how it worked
>> out.
>>
>> Alex
>>
>> --
>> Dr. Alex Singer
>> C.H. Best Institute
>> 112 College St. Room 70
>> University of Toronto
>> Toronto, Canada, M5G 1L6
>> 416-978-4033
>



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] strange density

[Jeffrey D Brodin]

Alex,

I modeled in the bis-tris with the tertiary amine and and his  
imidazole coordinating axially and the four oxygens coordinating in  
the equatorial plane. However, it's hard for me to tell from your  
images if there are two His coordinating? Either way, that crescent  
shape could easily be explained by a bis-tris molecule, you'll just  
have to figure out how exactly to model it in. It's also possible that  
the metal is a Mg, but as people have already mentioned, nitrogens  
probably wouldn't coordinate very tightly to a hard metal. Lastly, I'm  
also not sure off the top of my head how tightly bis-tris binds  
metals, but it should be an easy number to look up. Hope this helps,


Jeff
On Feb 24, 2011, at 9:02 AM, Alex Singer wrote:


Hi -- thank you for all your help.  The majority opinion seems to be a
metal for the sphere (Ni from the Ni-affinity column, which (Joe
Patel, correct) was used during purification, but Zn and Fe were also
mentioned), and either water molecules, bis-tris or some other small
molecule forming the crescent.  Just looking at the density, the
occupancy would seem to be quite high, so I'm surprised that a Ni ion
(or a contaminating metal ion) could have gone through the
purification and still remained at high enough concentration to be
clearly visible in the crystals.  However, I'll still try this but
first some points of clarification and questions which you can either
email me seperately or post to the the group.

a.  it was collected at beamline 19-BM at Argonne, so radiation damage
is an issue.  Thierry Fishmann -- for the gln residue, there was
difference density for the gamma carbon after the first conformation
was modeled in, thus the addition of the second conformation, which I
agree is suspect.  What does the radiation damage do chemically and
would that make the gamma carbon more mobile?

b.  Jeffrey D Brodin -- how did you model in the bis-tris?  Looking at
the bis-tris molecule from Hic-up, was the N at the centre of the
crescent and the O6 and O8 at the edges?

c.  JR Helliwell -- there are 4 molecules in the AU, but two H138's
are pointing into the solvent.  Thus the molar ratio of protein
molecules to "thing 1" is 4:1.  Also looking at the images, I see no
ice rings -- the images look pretty good.  Can you tell me more about
the series termination effects?

Again thank you for your help and I'll let the group know how it  
worked out.


Alex

--
Dr. Alex Singer
C.H. Best Institute
112 College St. Room 70
University of Toronto
Toronto, Canada, M5G 1L6
416-978-4033

Re: [ccp4bb] strange density

[John R Helliwell]

Dear Alex,
Sorry yes I was focussing on the crescent density, since I imagined
the metal atom assignment was secure. SInce Zn and Fe are now
mentioned you would need to consider a tuned Xray wavelength anomalous
experiment to be sure it is nickel, although the circumstatial
evidence looks promising already.

Re the series termination effects; see eg:-
A. Minichino, J. Habash, J. Raftery and J.R. Helliwell “The properties
of (2Fo-Fc) and (Fo-Fc) electron-density maps at medium-to-high
resolutions” (2003) Acta Cryst D59, 843-849.
and references therein.
That said my main idea of a cause of the crescent density being due to
severe ice rings is not the case, as you now describe, so I am not
sure what causes what you have there around your nickel ions.

Greetings,
John

On Thu, Feb 24, 2011 at 5:02 PM, Alex Singer
 wrote:
> Hi -- thank you for all your help.  The majority opinion seems to be a metal
> for the sphere (Ni from the Ni-affinity column, which (Joe Patel, correct)
> was used during purification, but Zn and Fe were also mentioned), and either
> water molecules, bis-tris or some other small molecule forming the crescent.
>  Just looking at the density, the occupancy would seem to be quite high, so
> I'm surprised that a Ni ion (or a contaminating metal ion) could have gone
> through the purification and still remained at high enough concentration to
> be clearly visible in the crystals.  However, I'll still try this but first
> some points of clarification and questions which you can either email me
> seperately or post to the the group.
>
> a.  it was collected at beamline 19-BM at Argonne, so radiation damage is an
> issue.  Thierry Fishmann -- for the gln residue, there was difference
> density for the gamma carbon after the first conformation was modeled in,
> thus the addition of the second conformation, which I agree is suspect.
>  What does the radiation damage do chemically and would that make the gamma
> carbon more mobile?
>
> b.  Jeffrey D Brodin -- how did you model in the bis-tris?  Looking at the
> bis-tris molecule from Hic-up, was the N at the centre of the crescent and
> the O6 and O8 at the edges?
>
> c.  JR Helliwell -- there are 4 molecules in the AU, but two H138's are
> pointing into the solvent.  Thus the molar ratio of protein molecules to
> "thing 1" is 4:1.  Also looking at the images, I see no ice rings -- the
> images look pretty good.  Can you tell me more about the series termination
> effects?
>
> Again thank you for your help and I'll let the group know how it worked out.
>
> Alex
>
> --
> Dr. Alex Singer
> C.H. Best Institute
> 112 College St. Room 70
> University of Toronto
> Toronto, Canada, M5G 1L6
> 416-978-4033
>



-- 
Professor John R Helliwell DSc

[ccp4bb] strange density

[Alex Singer]

Hi -- thank you for all your help.  The majority opinion seems to be a  
metal for the sphere (Ni from the Ni-affinity column, which (Joe  
Patel, correct) was used during purification, but Zn and Fe were also  
mentioned), and either water molecules, bis-tris or some other small  
molecule forming the crescent.  Just looking at the density, the  
occupancy would seem to be quite high, so I'm surprised that a Ni ion  
(or a contaminating metal ion) could have gone through the  
purification and still remained at high enough concentration to be  
clearly visible in the crystals.  However, I'll still try this but  
first some points of clarification and questions which you can either  
email me seperately or post to the the group.


a.  it was collected at beamline 19-BM at Argonne, so radiation damage  
is an issue.  Thierry Fishmann -- for the gln residue, there was  
difference density for the gamma carbon after the first conformation  
was modeled in, thus the addition of the second conformation, which I  
agree is suspect.  What does the radiation damage do chemically and  
would that make the gamma carbon more mobile?


b.  Jeffrey D Brodin -- how did you model in the bis-tris?  Looking at  
the bis-tris molecule from Hic-up, was the N at the centre of the  
crescent and the O6 and O8 at the edges?


c.  JR Helliwell -- there are 4 molecules in the AU, but two H138's  
are pointing into the solvent.  Thus the molar ratio of protein  
molecules to "thing 1" is 4:1.  Also looking at the images, I see no  
ice rings -- the images look pretty good.  Can you tell me more about  
the series termination effects?


Again thank you for your help and I'll let the group know how it worked out.

Alex

--
Dr. Alex Singer
C.H. Best Institute
112 College St. Room 70
University of Toronto
Toronto, Canada, M5G 1L6
416-978-4033

Re: [ccp4bb] strange density

[Dirk Kostrewa]

An additional information would be to calculate an anomalous difference 
map: at typical synchrotron wavelenght of ~1 A, Ni would have ~2 
electrons f'', whereas Cl would have only ~0.2 electrons f''. So, a 
strong anomalous difference density peak would be more consistent with 
Ni than with Cl.
However, if the data set was collected on a home source with CuKalpha 
wavelength, such a map would be not be very helpful here, because the Ni 
signal is about as weak as the Cl signal.


Best regards,

Dirk.

Am 24.02.11 16:37, schrieb Gloria Borgstahl:

I'm voting with Roger this time.
If I were you I would model a nickel in there (unless you have a 
better candidate)
if it is right, the distances to the His should be like seen in a SOD 
active site.
Then you can model the bonds and waters.  You may need partial 
occupancy on the metal.
Reminds me of the time I found a 6th water in MnSOD as I was 
making the token electron denisty
figure of the active site.  Found myself doing more refinement and a 
much better paper.


On Thu, Feb 24, 2011 at 8:15 AM, Roger Rowlett > wrote:


This looks suspiciously like a metal ion with 3 or more resolved
water ligands. It's hard to tell from the images provided, but it
looks like the metal could be close to octahedral, with 2 His and
3 water ligands well-defined. The estimated metal-nitrogen bond
distances might help narrow down the metal ion. For example,
tetrahedral Zn(II) has a Zn-N bond distance of around 2.0 A. I
suppose this could be a Mg(II) ion, since that is in your
crystallization mix, but I think that Mg(II) typically prefers
harder protein  ligands, e.g., carboxylates. Zn(II) is a very
common contaminant in solution, however, and should not
necessarily be discounted; indeed Zn(II) and other first row
transition metal ions would be expected to have a reasonable
affinity for medium hard/soft ligands such as vicinial His residues.

Cheers.

On 2/23/2011 7:34 PM, Alex Singer wrote:


cocrystallized in 2.5mM Glycero-3Phosphocholine and cryoprotected
by dipping in 
-- 


Roger S. Rowlett
Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu 




--

***
Dirk Kostrewa
Gene Center Munich, A5.07
Department of Biochemistry
Ludwig-Maximilians-Universität München
Feodor-Lynen-Str. 25
D-81377 Munich
Germany
Phone:  +49-89-2180-76845
Fax:+49-89-2180-76999
E-mail: kostr...@genzentrum.lmu.de
WWW:www.genzentrum.lmu.de
***

Re: [ccp4bb] strange density

[Gloria Borgstahl]

I'm voting with Roger this time.
If I were you I would model a nickel in there (unless you have a better
candidate)
if it is right, the distances to the His should be like seen in a SOD active
site.
Then you can model the bonds and waters.  You may need partial occupancy on
the metal.

Reminds me of the time I found a 6th water in MnSOD as I was making the
token electron denisty
figure of the active site.  Found myself doing more refinement and a much
better paper.

On Thu, Feb 24, 2011 at 8:15 AM, Roger Rowlett  wrote:

> This looks suspiciously like a metal ion with 3 or more resolved water
> ligands. It's hard to tell from the images provided, but it looks like the
> metal could be close to octahedral, with 2 His and 3 water ligands
> well-defined. The estimated metal-nitrogen bond distances might help narrow
> down the metal ion. For example, tetrahedral Zn(II) has a Zn-N bond distance
> of around 2.0 A. I suppose this could be a Mg(II) ion, since that is in your
> crystallization mix, but I think that Mg(II) typically prefers harder
> protein  ligands, e.g., carboxylates. Zn(II) is a very common contaminant in
> solution, however, and should not necessarily be discounted; indeed Zn(II)
> and other first row transition metal ions would be expected to have a
> reasonable affinity for medium hard/soft ligands such as vicinial His
> residues.
>
> Cheers.
>
> On 2/23/2011 7:34 PM, Alex Singer wrote:
>
>
> cocrystallized in 2.5mM Glycero-3Phosphocholine and cryoprotected by
> dipping in
>
> --
> --
> Roger S. Rowlett
> Professor
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
>
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@colgate.edu
>

Re: [ccp4bb] strange density

[Roger Rowlett]

  
  
This looks suspiciously like a metal ion with 3
  or more resolved water ligands. It's hard to tell from the
images provided, but it looks like the metal could be close to
octahedral, with 2 His and 3 water ligands well-defined. The
estimated metal-nitrogen bond distances might help narrow down the
metal ion. For example, tetrahedral Zn(II) has a Zn-N bond distance
of around 2.0 A. I suppose this could be a Mg(II) ion, since that is
in your crystallization mix, but I think that Mg(II) typically
prefers harder protein  ligands, e.g., carboxylates. Zn(II) is a
very common contaminant in solution, however, and should not
necessarily be discounted; indeed Zn(II) and other first row
transition metal ions would be expected to have a reasonable
affinity for medium hard/soft ligands such as vicinial His residues.

Cheers.

On 2/23/2011 7:34 PM, Alex Singer wrote:

  cocrystallized in 2.5mM Glycero-3Phosphocholine and cryoprotected
  by dipping in

-- 
  

Roger S. Rowlett
Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
  

  

Re: [ccp4bb] strange density

[Jrh]

Dear Alex 
I take it this density effect is in each of the four molecules?
I wonder if the crescent is a series termination effect, although relatively 
rare in our field. If so what might have caused it? Do you have (radial) gaps 
in your data set eg due to heavy ice rings?
Best wishes,
John

Prof John R Helliwell DSc


On 24 Feb 2011, at 00:34, Alex Singer  wrote:

> Hi -- I have a high resolution structure (1.6 A) where I'm ready to deposit 
> except I have
> some very strange density, shown in the two pictures here -- sort of a sphere 
> with a
> split cresent around it, falling between molecule A and B His 138 imidazole 
> rings.  The
> sphere is modeled as a Cl atom, more for "kicks" because resulting 2Fo-Fc 
> maps still have
> considerable positive difference density throughout the sphere.  There are 4 
> molecules in
> the AU, the imidazole ring of H138 in molecules C and D point into a solvent 
> channel.
> Crystallization conditions are 0.2M Mg Chloride, 0.1M Bis-Tris pH6.5, 25% 
> PEG3350,
> cocrystallized in 2.5mM Glycero-3Phosphocholine and cryoprotected by dipping 
> in
> Paratone_N oil.
> 
> Let me know what you're thoughts are and thank you for your help.
> 
> Alex Singer
> 
> 
> -- 
> Dr. Alex Singer
> C.H. Best Institute
> 112 College St. Room 70
> University of Toronto
> Toronto, Canada, M5G 1L6
> 416-978-4033
> 
> 

Re: [ccp4bb] strange density

[Patel, Joe]

Hi Alex,

Was it purified via a Ni2+ resin?  Is the protein oligomeric in
solution?  Could it have stripped an ion out during purification and
brought it all the way through to crystallisation?

Have you tried refining a Ni2+ in the location?

JP


--
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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Alex Singer
Sent: 24 February 2011 00:35
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] strange density

Hi -- I have a high resolution structure (1.6 A) where I'm ready to
deposit except I have some very strange density, shown in the two
pictures here -- sort of a sphere with a split cresent around it,
falling between molecule A and B His 138 imidazole rings.  The sphere is
modeled as a Cl atom, more for "kicks" because resulting 2Fo-Fc maps
still have considerable positive difference density throughout the
sphere.  There are 4 molecules in the AU, the imidazole ring of H138 in
molecules C and D point into a solvent channel.
Crystallization conditions are 0.2M Mg Chloride, 0.1M Bis-Tris pH6.5,
25% PEG3350, cocrystallized in 2.5mM Glycero-3Phosphocholine and
cryoprotected by dipping in Paratone_N oil.

Let me know what you're thoughts are and thank you for your help.

Alex Singer


--
Dr. Alex Singer
C.H. Best Institute
112 College St. Room 70
University of Toronto
Toronto, Canada, M5G 1L6
416-978-4033

Re: [ccp4bb] Strange density on Serine oxygen.

[Vinson LIANG]

Hi Linda and all, 

Thank you very much for your suggestion. 

I just help to solve the structure of this protein. And I'm not sure if the 
protein is properly sequenced first. I'll confirm the sequence first. I'll let 
you know if it is not the sequence's problem. 


I very appreciate help from all you guys, so quick and helpful. 

Best wishes, 

Vinson 





发件人： Linda Schuldt 
收件人： CCP4BB@JISCMAIL.AC.UK
发送日期： 2010/11/24 (周三) 9:35:33 下午
主 题： Re: [ccp4bb] Strange density on Serine oxygen.

Hi Vinson,

along these lines: did you check the molecular weight of your protein with
MS? This should help to answer if the molecular weight deviates from the
expected one.

Best wishes,
Linda

Savvas Savvides schrieb:
> Hi Vinson
> Beyond the possibility for another type of residue as already suggested by
> Phil and Mark, there is also the possibility of O-linked glycosylation of
> the serine and threonine, if your protein undergoes such
> post-translational modification and it has been expressed via an
> expression system that processes the protein in that way.
> Ser/Thr tandems are well known targets for O-glycosylation
> (http://www.cbs.dtu.dk/databases/OGLYCBASE/).
>
> best regards
> Savvas
>
> 
> Savvas Savvides
> Unit for Structural Biology @ L-ProBE
> Ghent University
> K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
> Ph. +32  (0)472 928 519 http://www.LProBE.ugent.be/xray.html
>
>
>
> On 24 Nov 2010, at 13:10, Vinson LIANG wrote:
>
>> Dear all,
>>
>> I'm refining a structure and find some strange triangle density on the
>> oxygen of Ser and Thr at the C terminus. One picture of the strange
>> density is attached here. Could anyone please give me some suggestions
>> on what this could be?
>>
>> The buffer used during purification is PBS, Tris and NaCl. And
>> crystallization condition contains PEG3,350 and Mg(NO3)2.
>>
>> Thank you all in advance for any suggestion.
>>
>> Best,
>>
>> Vinson Liang
>>
>>
>>
>>  
>
>



  

Re: [ccp4bb] Strange density on Serine oxygen.

[Vinson LIANG]

Hi Savvas, 

You're right. It shouldn't be O-glycosylation, since the protein is from 
Archaea 
and expressed in E. coli. Most possiblly, I think it's something wrong with the 
sequence.

Thanks for your help, 

Best, 

Vinson


 




发件人： Savvas Savvides 
收件人： Vinson LIANG 
抄 送： CCP4BB@JISCMAIL.AC.UK
发送日期： 2010/11/24 (周三) 9:48:28 下午
主 题： Re: [ccp4bb] Strange density on Serine oxygen.

Hi Vinson 
is O-glycosylation possible at all given the origin of the protein and the 
expression system used? 
best 
Savvas





On 24 Nov 2010, at 14:42, Vinson LIANG wrote:

Dear Savas and all,
>
>
>Thank you very much for all your quick suggestions.
>
>I have tried Tyr and it turns out to fit the density very well. I will have 
>the 
>protein sequenced again to see if it is wrong sequece or O-linked 
>glycosylation.
>
>I'll let you know if it turns out to be O-linked glycosylation.
>
>All the best,
>
>Vinson 
>
>

发件人： Savvas Savvides 
>收件人： Vinson LIANG 
>抄 送： ccp...@jiscmail.ac.uk
>发送日期： 2010/11/24 (周三) 9:27:31 下午
>主 题： Re: [ccp4bb] Strange density on Serine oxygen.
>
>Hi Vinson 
>Beyond the possibility for another type of residue as already suggested by 
>Phil 
>and Mark, there is also the possibility of O-linked glycosylation of the 
>serine 
>and threonine, if your protein undergoes such post-translational modification 
>and it has been expressed via an expression system that processes the protein 
>in 
>that way.
>Ser/Thr tandems are well known targets for O-glycosylation 
>(http://www.cbs.dtu.dk/databases/OGLYCBASE/).
>
>
>best regards
>Savvas
>
>
>
>Savvas Savvides
>Unit for Structural Biology @ L-ProBE
>Ghent University
>K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
>Ph. +32  (0)472 928 519 http://www.LProBE.ugent.be/xray.html
>
>
>
>
>On 24 Nov 2010, at 13:10, Vinson LIANG wrote:
>
>Dear all,
>>
>>I'm refining a structure and find some strange triangle density on the oxygen 
>>of 
>>Ser and Thr at the C terminus. One picture of the strange density is attached 
>>here. Could anyone please give me some suggestions on what this could be?
>>
>>The buffer used during purification is PBS, Tris and NaCl. And 
>>crystallization 
>>condition contains PEG3,350 and Mg(NO3)2.
>>
>>Thank you all in advance for any suggestion.
>>
>>Best,
>>
>>Vinson Liang
>>
>>
>> 
>
> 
> 



  

Re: [ccp4bb] Strange density on Serine oxygen.

[Herman . Schreuder]

This density does not look at all like O-glycosylation. 
Best regards,
Herman




From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of 
Savvas Savvides
Sent: Wednesday, November 24, 2010 2:48 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density on Serine oxygen.


Hi Vinson 
is O-glycosylation possible at all given the origin of the protein and 
the expression system used? 
best 
Savvas






On 24 Nov 2010, at 14:42, Vinson LIANG wrote:



Dear Savas and all,
 
Thank you very much for all your quick suggestions.
 
I have tried Tyr and it turns out to fit the density very well. 
I will have the protein sequenced again to see if it is wrong sequece or 
O-linked glycosylation.
 
I'll let you know if it turns out to be O-linked glycosylation.
 
All the best,
 
Vinson 




发件人： Savvas Savvides 
收件人： Vinson LIANG 
抄 送： CCP4BB@JISCMAIL.AC.UK
发送日期： 2010/11/24 (周三) 9:27:31 下午
主 题： Re: [ccp4bb] Strange density on Serine oxygen.

Hi Vinson 
Beyond the possibility for another type of residue as already 
suggested by Phil and Mark, there is also the possibility of O-linked 
glycosylation of the serine and threonine, if your protein undergoes such 
post-translational modification and it has been expressed via an expression 
system that processes the protein in that way.
Ser/Thr tandems are well known targets for O-glycosylation 
(http://www.cbs.dtu.dk/databases/OGLYCBASE/).

best regards
Savvas



Savvas Savvides
Unit for Structural Biology @ L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Ph. +32  (0)472 928 519 http://www.LProBE.ugent.be/xray.html



On 24 Nov 2010, at 13:10, Vinson LIANG wrote:



Dear all,
 
I'm refining a structure and find some strange triangle 
density on the oxygen of Ser and Thr at the C terminus. One picture of the 
strange density is attached here. Could anyone please give me some suggestions 
on what this could be?
 
The buffer used during purification is PBS, Tris and 
NaCl. And crystallization condition contains PEG3,350 and Mg(NO3)2.
 
Thank you all in advance for any suggestion.
 
Best,
 
Vinson Liang
 
 

 



 

 

Re: [ccp4bb] Strange density on Serine oxygen.

[Shekhar Mande]

Phosphoserine ?

शेखर चिं मांडे
हैदराबाद


On Wed, Nov 24, 2010 at 5:40 PM, Vinson LIANG
wrote:

> Dear all,
>
> I'm refining a structure and find some strange triangle density on the
> oxygen of Ser and Thr at the C terminus. One picture of the strange density
> is attached here. Could anyone please give me some suggestions on what this
> could be?
>
> The buffer used during purification is PBS, Tris and NaCl. And
> crystallization condition contains PEG3,350 and Mg(NO3)2.
>
> Thank you all in advance for any suggestion.
>
> Best,
>
> Vinson Liang
>
>
>
>
>



--

Re: [ccp4bb] Strange density on Serine oxygen.

[Savvas Savvides]

Hi Vinson
is O-glycosylation possible at all given the origin of the protein and the 
expression system used? 
best 
Savvas




On 24 Nov 2010, at 14:42, Vinson LIANG wrote:

> Dear Savas and all,
>  
> Thank you very much for all your quick suggestions.
>  
> I have tried Tyr and it turns out to fit the density very well. I will have 
> the protein sequenced again to see if it is wrong sequece or O-linked 
> glycosylation.
>  
> I'll let you know if it turns out to be O-linked glycosylation.
>  
> All the best,
>  
> Vinson 
> 发件人： Savvas Savvides 
> 收件人： Vinson LIANG 
> 抄 送： CCP4BB@JISCMAIL.AC.UK
> 发送日期： 2010/11/24 (周三) 9:27:31 下午
> 主 题： Re: [ccp4bb] Strange density on Serine oxygen.
> 
> Hi Vinson
> Beyond the possibility for another type of residue as already suggested by 
> Phil and Mark, there is also the possibility of O-linked glycosylation of the 
> serine and threonine, if your protein undergoes such post-translational 
> modification and it has been expressed via an expression system that 
> processes the protein in that way.
> Ser/Thr tandems are well known targets for O-glycosylation 
> (http://www.cbs.dtu.dk/databases/OGLYCBASE/).
> 
> best regards
> Savvas
> 
> 
> Savvas Savvides
> Unit for Structural Biology @ L-ProBE
> Ghent University
> K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
> Ph. +32  (0)472 928 519 http://www.LProBE.ugent.be/xray.html
> 
> 
> 
> On 24 Nov 2010, at 13:10, Vinson LIANG wrote:
> 
>> Dear all,
>>  
>> I'm refining a structure and find some strange triangle density on the 
>> oxygen of Ser and Thr at the C terminus. One picture of the strange density 
>> is attached here. Could anyone please give me some suggestions on what this 
>> could be?
>>  
>> The buffer used during purification is PBS, Tris and NaCl. And 
>> crystallization condition contains PEG3,350 and Mg(NO3)2.
>>  
>> Thank you all in advance for any suggestion.
>>  
>> Best,
>>  
>> Vinson Liang
>>  
>>  
>> 
>>  
> 
> 
>  
> 
>  

Re: [ccp4bb] Strange density on Serine oxygen.

[Linda Schuldt]

Hi Vinson,

along these lines: did you check the molecular weight of your protein with
MS? This should help to answer if the molecular weight deviates from the
expected one.

Best wishes,
Linda

Savvas Savvides schrieb:
> Hi Vinson
> Beyond the possibility for another type of residue as already suggested by
> Phil and Mark, there is also the possibility of O-linked glycosylation of
> the serine and threonine, if your protein undergoes such
> post-translational modification and it has been expressed via an
> expression system that processes the protein in that way.
> Ser/Thr tandems are well known targets for O-glycosylation
> (http://www.cbs.dtu.dk/databases/OGLYCBASE/).
>
> best regards
> Savvas
>
> 
> Savvas Savvides
> Unit for Structural Biology @ L-ProBE
> Ghent University
> K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
> Ph. +32  (0)472 928 519 http://www.LProBE.ugent.be/xray.html
>
>
>
> On 24 Nov 2010, at 13:10, Vinson LIANG wrote:
>
>> Dear all,
>>
>> I'm refining a structure and find some strange triangle density on the
>> oxygen of Ser and Thr at the C terminus. One picture of the strange
>> density is attached here. Could anyone please give me some suggestions
>> on what this could be?
>>
>> The buffer used during purification is PBS, Tris and NaCl. And
>> crystallization condition contains PEG3,350 and Mg(NO3)2.
>>
>> Thank you all in advance for any suggestion.
>>
>> Best,
>>
>> Vinson Liang
>>
>>
>>
>>  
>
>

Re: [ccp4bb] Strange density on Serine oxygen.

[Vinson LIANG]

Dear Savas and all, 


Thank you very much for all your quick suggestions. 

I have tried Tyr and it turns out to fit the density very well. I will have the 
protein sequenced again to see if it is wrong sequece or O-linked 
glycosylation. 


I'll let you know if it turns out to be O-linked glycosylation. 

All the best, 

Vinson 



发件人： Savvas Savvides 
收件人： Vinson LIANG 
抄 送： CCP4BB@JISCMAIL.AC.UK
发送日期： 2010/11/24 (周三) 9:27:31 下午
主 题： Re: [ccp4bb] Strange density on Serine oxygen.

Hi Vinson 
Beyond the possibility for another type of residue as already suggested by Phil 
and Mark, there is also the possibility of O-linked glycosylation of the serine 
and threonine, if your protein undergoes such post-translational modification 
and it has been expressed via an expression system that processes the protein 
in 
that way.
Ser/Thr tandems are well known targets for O-glycosylation 
(http://www.cbs.dtu.dk/databases/OGLYCBASE/).

best regards
Savvas



Savvas Savvides
Unit for Structural Biology @ L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Ph. +32  (0)472 928 519 http://www.LProBE.ugent.be/xray.html



On 24 Nov 2010, at 13:10, Vinson LIANG wrote:

Dear all,
>
>I'm refining a structure and find some strange triangle density on the oxygen 
>of 
>Ser and Thr at the C terminus. One picture of the strange density is attached 
>here. Could anyone please give me some suggestions on what this could be?
>
>The buffer used during purification is PBS, Tris and NaCl. And crystallization 
>condition contains PEG3,350 and Mg(NO3)2.
>
>Thank you all in advance for any suggestion.
>
>Best,
>
>Vinson Liang
>
>
> 


  

Re: [ccp4bb] Strange density on Serine oxygen.

[Savvas Savvides]

Hi Vinson
Beyond the possibility for another type of residue as already suggested by Phil 
and Mark, there is also the possibility of O-linked glycosylation of the serine 
and threonine, if your protein undergoes such post-translational modification 
and it has been expressed via an expression system that processes the protein 
in that way.
Ser/Thr tandems are well known targets for O-glycosylation 
(http://www.cbs.dtu.dk/databases/OGLYCBASE/).

best regards
Savvas


Savvas Savvides
Unit for Structural Biology @ L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Ph. +32  (0)472 928 519 http://www.LProBE.ugent.be/xray.html



On 24 Nov 2010, at 13:10, Vinson LIANG wrote:

> Dear all,
>  
> I'm refining a structure and find some strange triangle density on the oxygen 
> of Ser and Thr at the C terminus. One picture of the strange density is 
> attached here. Could anyone please give me some suggestions on what this 
> could be?
>  
> The buffer used during purification is PBS, Tris and NaCl. And 
> crystallization condition contains PEG3,350 and Mg(NO3)2.
>  
> Thank you all in advance for any suggestion.
>  
> Best,
>  
> Vinson Liang
>  
>  
> 
>  

Re: [ccp4bb] Strange density on Serine oxygen.

[Mark J van Raaij]

look like tyrosines to me!

Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia
c/Darwin 3, Campus Cantoblanco
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.researcherid.com/rid/B-3678-2009



On 24 Nov 2010, at 13:10, Vinson LIANG wrote:

> Dear all,
>  
> I'm refining a structure and find some strange triangle density on the oxygen 
> of Ser and Thr at the C terminus. One picture of the strange density is 
> attached here. Could anyone please give me some suggestions on what this 
> could be?
>  
> The buffer used during purification is PBS, Tris and NaCl. And 
> crystallization condition contains PEG3,350 and Mg(NO3)2.
>  
> Thank you all in advance for any suggestion.
>  
> Best,
>  
> Vinson Liang
>  
>  
> 
>  

Re: [ccp4bb] Strange density on Serine oxygen.

[Phil Evans]

Are you sure the sequence is right? It looks like tyrosine
Phil

On 24 Nov 2010, at 12:10, Vinson LIANG wrote:

> Dear all,
>  
> I'm refining a structure and find some strange triangle density on the oxygen 
> of Ser and Thr at the C terminus. One picture of the strange density is 
> attached here. Could anyone please give me some suggestions on what this 
> could be?
>  
> The buffer used during purification is PBS, Tris and NaCl. And 
> crystallization condition contains PEG3,350 and Mg(NO3)2.
>  
> Thank you all in advance for any suggestion.
>  
> Best,
>  
> Vinson Liang
>  
>  
> 
>  

Re: [ccp4bb] Strange Density

[Warren DeLano]

One of the great things about open-source code is that you simply have
no idea how, where, and when people will apply it.  Nevertheless, it is
important for everyone to recognize that the following activities are
*unsupported* uses of the PyMOL open-source software:

1. solid, isomesh, or dot rendering of religious density artifacts.
2. transubstantiation (e.g. wine -> blood, PBS -> HEPES).
3. reanimation of organic materials, be they ordinary or holy.
4. miracles and revelations (beyond your thesis project).
5. proselytization: whether print, animated, or interactive.
6. heresy, sacrilege, or apostasy, of all kinds, in all media,
worldwide.

USE OF PYMOL FOR ANY OR ALL OF THE ABOVE ACTIVITIES IS ON AN-AS IS BASIS
AND AT YOUR OWN RISK.  DELANO SCIENTIFIC LLC DISCLAIMS ALL WARRANTIES
AND LIABILITY FOR RETALIATORY ACTS OF A VENGEFUL GOD (OR GODS). IN NO
EVENT SHALL DELANO SCIENTIFIC LLC BE LIABLE FOR BOLTS OF LIGHTING,
EARTHQUAKES, TSUNAMIS, VOLCANIC ERUPTIONS, OR CIVILIZATION-ENDING
METEORITE IMPACTS ARISING OUT OF OR IN CONNECTION WITH UNSUPPORTED
APPLICATIONS OF THE PYMOL SOFTWARE(*).

* If you do choose to tempt fate, be sure to take a copy of the
source-code with you:  We hear that Hell's 666-BAUD internet connection
is woefully inadequate for pulling down those ever-so-bloated Coot and
PyMOL binaries (even if you do have an eternity to wait).

Thank you for your understanding and consideration.



From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On
Behalf Of Jesus Christiano
Sent: Monday, April 02, 2007 5:01 AM
To: CCP4BB@JISCMAIL.AC.UK
    Subject: [ccp4bb] Strange Density


Hello My Friends,

I hope you are all enjoying the Sabbath on this first day of
April.

I have recently been struggling to understand the meaning of my
structure and now I am looking for guidance. After a long period
aimlessly wandering through dense regions of ambiguity, I have of late
been inspired with profound clarity. 

First, I should enlighten you about my structure. I believe that
is one that promises great hope for all people. However, I have not been
without my detractors, for many have lost faith in its absolute value to
humanity. 

My question is about the nature of a strange and wonderful
positive density I have found. It seems to be coordinated by a trinity
of bonds--yet otherwise floats mystically in a heavenly fashion. I do
not fully understand it though somehow I know that it holds the key to
my perpetual happiness. 

I must confess that resurrecting this project has been trying. I
have been tempted to seek another path many times, often posessed with
thoughts of worldly acquisition. But if I can not see the light that
this density offers, a comittee will crucify me, rendering salvation of
my thesis difficult without divine intervention. Such a sacrifice I can
not make without grave consideration. 

I have put an image of this strange density here:

http://strangedensity.blogspot.com/

Please, if anyone can provide guidance, I will be eternally
grateful. 

Peace be with you,
Jesus H. Christiano 

Re: [ccp4bb] Strange Density

[Eleanor Dodson]

Superb picture - thankyou - will add it to the lab gallery!
Eleanor

William Scott wrote:

Hey, dude, thanks for writing!

I could swear (so to speak) You sat next to me on an 11 hour airplane
flight not too long ago and tried to strike up a similar conversation. I
apologize for being too engrossed in the latest Dawkins book.

However, we encountered a similar structural riddle in need of
interpretation recently.  Depicted on this link is our best guess at the
Transition-State structure.  I believe you will be especially taken by the
leaving group, although I cannot vouch for the orientation and its
subsequent trajectory:
http://xanana.ucsc.edu/scottlab/the_moment_of_creation.jpg

Hope this helps.

PS:  I had an undergraduate in the lab recently who was working on a
homologue to your project.  Unfortunately he was denied entry to graduate
school.  His grades were quite good, but I hear he got nailed on the
boards.



Jesus Christiano wrote:
  

Hello My Friends,

I hope you are all enjoying the Sabbath on this first day of April.

I have recently been struggling to understand the meaning of my structure
and now I am looking for guidance. After a long period aimlessly wandering
through dense regions of ambiguity, I have of late been inspired with
profound clarity.

First, I should enlighten you about my structure. I believe that is one
that
promises great hope for all people. However, I have not been without my
detractors, for many have lost faith in its absolute value to humanity.

My question is about the nature of a strange and wonderful positive
density
I have found. It seems to be coordinated by a trinity of bonds--yet
otherwise floats mystically in a heavenly fashion. I do not fully
understand
it though somehow I know that it holds the key to my perpetual happiness.

I must confess that resurrecting this project has been trying. I have been
tempted to seek another path many times, often posessed with thoughts of
worldly acquisition. But if I can not see the light that this density
offers, a comittee will crucify me, rendering salvation of my thesis
difficult without divine intervention. Such a sacrifice I can not make
without grave consideration.

I have put an image of this strange density here:

http://strangedensity.blogspot.com/

Please, if anyone can provide guidance, I will be eternally grateful.

Peace be with you,
Jesus H. Christiano





  

Re: [ccp4bb] Strange Density

[William Scott]

Hey, dude, thanks for writing!

I could swear (so to speak) You sat next to me on an 11 hour airplane
flight not too long ago and tried to strike up a similar conversation. I
apologize for being too engrossed in the latest Dawkins book.

However, we encountered a similar structural riddle in need of
interpretation recently.  Depicted on this link is our best guess at the
Transition-State structure.  I believe you will be especially taken by the
leaving group, although I cannot vouch for the orientation and its
subsequent trajectory:
http://xanana.ucsc.edu/scottlab/the_moment_of_creation.jpg

Hope this helps.

PS:  I had an undergraduate in the lab recently who was working on a
homologue to your project.  Unfortunately he was denied entry to graduate
school.  His grades were quite good, but I hear he got nailed on the
boards.



Jesus Christiano wrote:
> Hello My Friends,
>
> I hope you are all enjoying the Sabbath on this first day of April.
>
> I have recently been struggling to understand the meaning of my structure
> and now I am looking for guidance. After a long period aimlessly wandering
> through dense regions of ambiguity, I have of late been inspired with
> profound clarity.
>
> First, I should enlighten you about my structure. I believe that is one
> that
> promises great hope for all people. However, I have not been without my
> detractors, for many have lost faith in its absolute value to humanity.
>
> My question is about the nature of a strange and wonderful positive
> density
> I have found. It seems to be coordinated by a trinity of bonds--yet
> otherwise floats mystically in a heavenly fashion. I do not fully
> understand
> it though somehow I know that it holds the key to my perpetual happiness.
>
> I must confess that resurrecting this project has been trying. I have been
> tempted to seek another path many times, often posessed with thoughts of
> worldly acquisition. But if I can not see the light that this density
> offers, a comittee will crucify me, rendering salvation of my thesis
> difficult without divine intervention. Such a sacrifice I can not make
> without grave consideration.
>
> I have put an image of this strange density here:
>
> http://strangedensity.blogspot.com/
>
> Please, if anyone can provide guidance, I will be eternally grateful.
>
> Peace be with you,
> Jesus H. Christiano
>

[ccp4bb] Strange Density

[Jesus Christiano]

Hello My Friends,

I hope you are all enjoying the Sabbath on this first day of April.

I have recently been struggling to understand the meaning of my structure
and now I am looking for guidance. After a long period aimlessly wandering
through dense regions of ambiguity, I have of late been inspired with
profound clarity.

First, I should enlighten you about my structure. I believe that is one that
promises great hope for all people. However, I have not been without my
detractors, for many have lost faith in its absolute value to humanity.

My question is about the nature of a strange and wonderful positive density
I have found. It seems to be coordinated by a trinity of bonds--yet
otherwise floats mystically in a heavenly fashion. I do not fully understand
it though somehow I know that it holds the key to my perpetual happiness.

I must confess that resurrecting this project has been trying. I have been
tempted to seek another path many times, often posessed with thoughts of
worldly acquisition. But if I can not see the light that this density
offers, a comittee will crucify me, rendering salvation of my thesis
difficult without divine intervention. Such a sacrifice I can not make
without grave consideration.

I have put an image of this strange density here:

http://strangedensity.blogspot.com/

Please, if anyone can provide guidance, I will be eternally grateful.

Peace be with you,
Jesus H. Christiano