Re: [ccp4bb] pseudo-centering or twinning problem in P3

[Diana Tomchick]

Maybe, but it may also indicate that the problem isn’t twinning, but simply 
pseudo symmetry.

Zanuda is one way to double-check, and it takes approximately a day to 
re-process the data, run the MR and run Zanuda.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Aug 8, 2017, at 1:27 PM, Eleanor Dodson 
> wrote:

That seems hard work to me! Data quality suffers when you discard multiplicity..
 Eleanor

On 8 August 2017 at 17:15, Sudipta Bhattacharyya 
>
 wrote:
Hi Giorgio,

I also suspect the presence of twining and as Eleanor has suggested the L test 
is the best test to detect that (if also t-NCS is not present). You can 
reprocess your data in P1, do MR in P1 and then feed your P1 model and mtz file 
to zanuda to see what SG it suggests. That trick once worked for me.

Cheers,
Sudipta.


Sudipta Bhattacharyya,
Postdoctoral fellow
Department of Molecular Biosciences
University of Texas at Austin
Texas, USA.


On Tue, Aug 8, 2017 at 8:27 AM, Eleanor Dodson 
> wrote:
First of all - check whether your crystal data shows twinning - thee is an 
L-test plot which usually gives a clear indication and if you are using GUI2 
the report suggests if this is so.


If you have twinning the most likely SG is P31 (or P32 ) - you cant tell the 
difference till the structure is solved.

Check the merging stats in that point group -
Then try MR searches in the two spacegroups.

The C2 and I2 SGs are probably sub-group possibilities if you reindex - there 
are common cell dimensions -pointless output tells you how to do that - but in 
general one always chooses the highest symmetry that gives reasonable merging 
statistics and is sensible.

Eleanor



On 8 August 2017 at 14:15, Giorgio Giardina 
> wrote:
Hello everybody,

I think I have a pseudo-centering problem.

I have a 1.6 ang. dataset of a mutant  protein that is an homodimer in solution.
Data processing gives the following SG:

Space group: P 31 2 1
Average unit cell:  111.36  111.36   28.56   90.00   90.00  120.00
Average mosaicity:   0.24
Rmerge Overall 0.07

However with this SG the unit Cell is too small and monomer doesn't fit the in 
the AU.

I reprocessed the data in other possible SG (including P6) and finally I got 2 
equivalent SG's in which I can get a correct Molecular Replacement solution:

Space group: I 1 2 1
Average unit cell:   57.07  111.11  192.90   90.00   90.07   90.00

and

Space group: C 1 2 1
Average unit cell:  201.10  111.11   57.07   90.00  106.42   90.00

Both with Average mosaicity:   0.38 and Rmerge Overall 0.06,
but the corresponding MR solutions do not refine, with R-factor stuck to 45-47%.

From what I understand, in the I121 SG I have the NC two-fold axis of the dimer 
at 1/2a and this originates the pseudo centering and the small P3 Cell

Largest Patterson peak with length larger than 15 Angstrom:
 Frac. coord.  :0.5000.0000.000
 Distance to origin:   28.566
 Height relative to origin :   53.830 %

I'm really not so good with symmetry, so I'll be grateful for any 
suggestion/help/solution from you out there.
Many thanks,
Giorgio







UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] pseudo-centering or twinning problem in P3

[Eleanor Dodson]

That seems hard work to me! Data quality suffers when you discard
multiplicity..
 Eleanor

On 8 August 2017 at 17:15, Sudipta Bhattacharyya <
sudiptabhattacharyya.iit...@gmail.com> wrote:

> Hi Giorgio,
>
> I also suspect the presence of twining and as Eleanor has suggested the L
> test is the best test to detect that (if also t-NCS is not present). You
> can reprocess your data in P1, do MR in P1 and then feed your P1 model and
> mtz file to zanuda to see what SG it suggests. That trick once worked for
> me.
>
> Cheers,
> Sudipta.
>
>
> Sudipta Bhattacharyya,
> Postdoctoral fellow
> Department of Molecular Biosciences
> University of Texas at Austin
> Texas, USA.
>
>
> On Tue, Aug 8, 2017 at 8:27 AM, Eleanor Dodson 
> wrote:
>
>> First of all - check whether your crystal data shows twinning - thee is
>> an L-test plot which usually gives a clear indication and if you are using
>> GUI2 the report suggests if this is so.
>>
>>
>> If you have twinning the most likely SG is P31 (or P32 ) - you cant tell
>> the difference till the structure is solved.
>>
>> Check the merging stats in that point group -
>> Then try MR searches in the two spacegroups.
>>
>> The C2 and I2 SGs are probably sub-group possibilities if you reindex -
>> there are common cell dimensions -pointless output tells you how to do that
>> - but in general one always chooses the highest symmetry that gives
>> reasonable merging statistics and is sensible.
>>
>> Eleanor
>>
>>
>>
>> On 8 August 2017 at 14:15, Giorgio Giardina > > wrote:
>>
>>> Hello everybody,
>>>
>>> I think I have a pseudo-centering problem.
>>>
>>> I have a 1.6 ang. dataset of a mutant  protein that is an homodimer in
>>> solution.
>>> Data processing gives the following SG:
>>>
>>> Space group: P 31 2 1
>>> Average unit cell:  111.36  111.36   28.56   90.00   90.00  120.00
>>> Average mosaicity:   0.24
>>> Rmerge Overall 0.07
>>>
>>> However with this SG the unit Cell is too small and monomer doesn't fit
>>> the in the AU.
>>>
>>> I reprocessed the data in other possible SG (including P6) and finally I
>>> got 2 equivalent SG's in which I can get a correct Molecular Replacement
>>> solution:
>>>
>>> Space group: I 1 2 1
>>> Average unit cell:   57.07  111.11  192.90   90.00   90.07   90.00
>>>
>>> and
>>>
>>> Space group: C 1 2 1
>>> Average unit cell:  201.10  111.11   57.07   90.00  106.42   90.00
>>>
>>> Both with Average mosaicity:   0.38 and Rmerge Overall 0.06,
>>> but the corresponding MR solutions do not refine, with R-factor stuck to
>>> 45-47%.
>>>
>>> From what I understand, in the I121 SG I have the NC two-fold axis of
>>> the dimer at 1/2a and this originates the pseudo centering and the small P3
>>> Cell
>>>
>>> Largest Patterson peak with length larger than 15 Angstrom:
>>>  Frac. coord.  :0.5000.0000.000
>>>  Distance to origin:   28.566
>>>  Height relative to origin :   53.830 %
>>>
>>> I'm really not so good with symmetry, so I'll be grateful for any
>>> suggestion/help/solution from you out there.
>>> Many thanks,
>>> Giorgio
>>>
>>
>>
>

Re: [ccp4bb] doubt regarding setting resolution cutoff

[Eleanor Dodson]

You have some horrible ice rings - some data processing software may be
able to cut them out.. how are you processing it?
Eleanor

On 8 August 2017 at 15:43, Christian Roth 
wrote:

> Your plots look strangely different to the old Scala output you posted
> before, but never mind.
>
> Paul is right that a negative intensity is not desired and your crystal
> has some issues with ice.
>
> That one icering around 2.26 must be massive taken into account how
> haywire your curve goes there.
>
> Have you had a look at the images? There should be something visible in
> that area.
>
> Christian
>
> Am 08.08.2017 um 15:17 schrieb Paul Emsley:
>
> On 08/08/2017 15:07, Satvik Kumar wrote:
>
> Dear Prof. Powell and Prof. Dodson,
>
> Thanks for your reply and advise.
>
> As per your suggestion, I have re-scaled the intensities using Aimless at
> 1.861 A.
>
> I observe that the I/sigI has dropped to -0.8
>
>
> That's not good.
>
> > and the behaviour of CC_1/2 is still anomalous.
>
> That made me laugh out loud. Perhaps not the best choice of adjective.
>
>
> Also, when I inspect the Wilson plot (Fig. 1), I observe that the curve
> does not fall smoothly with respect to the reference curve (blue). Even
> with respect to one more Wilson plot from CCP4 website (Fig. 2), the curve
> from my aimless output is different and discontinuous.
>
>
> Icy!
>
> /me wonders if CCP4 are distributing auspex yet...
>
>
> The second moment of I is constant only up to a resolution of 2.4 Å at a
> value of 3 (Fig. 3). I was not able to get some other plot to compare
> against mine.
>
> Please tell me if I can still go ahead and refine at 1.861 A.
>
>
> No you can't.
>
> Maybe with some chopping you can rescue some reflections beyond 2.1.
>
> Paul
>
>
>

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Re: [ccp4bb] pseudo-centering or twinning problem in P3

[Sudipta Bhattacharyya]

Hi Giorgio,

I also suspect the presence of twining and as Eleanor has suggested the L
test is the best test to detect that (if also t-NCS is not present). You
can reprocess your data in P1, do MR in P1 and then feed your P1 model and
mtz file to zanuda to see what SG it suggests. That trick once worked for
me.

Cheers,
Sudipta.


Sudipta Bhattacharyya,
Postdoctoral fellow
Department of Molecular Biosciences
University of Texas at Austin
Texas, USA.


On Tue, Aug 8, 2017 at 8:27 AM, Eleanor Dodson 
wrote:

> First of all - check whether your crystal data shows twinning - thee is an
> L-test plot which usually gives a clear indication and if you are using
> GUI2 the report suggests if this is so.
>
>
> If you have twinning the most likely SG is P31 (or P32 ) - you cant tell
> the difference till the structure is solved.
>
> Check the merging stats in that point group -
> Then try MR searches in the two spacegroups.
>
> The C2 and I2 SGs are probably sub-group possibilities if you reindex -
> there are common cell dimensions -pointless output tells you how to do that
> - but in general one always chooses the highest symmetry that gives
> reasonable merging statistics and is sensible.
>
> Eleanor
>
>
>
> On 8 August 2017 at 14:15, Giorgio Giardina 
> wrote:
>
>> Hello everybody,
>>
>> I think I have a pseudo-centering problem.
>>
>> I have a 1.6 ang. dataset of a mutant  protein that is an homodimer in
>> solution.
>> Data processing gives the following SG:
>>
>> Space group: P 31 2 1
>> Average unit cell:  111.36  111.36   28.56   90.00   90.00  120.00
>> Average mosaicity:   0.24
>> Rmerge Overall 0.07
>>
>> However with this SG the unit Cell is too small and monomer doesn't fit
>> the in the AU.
>>
>> I reprocessed the data in other possible SG (including P6) and finally I
>> got 2 equivalent SG's in which I can get a correct Molecular Replacement
>> solution:
>>
>> Space group: I 1 2 1
>> Average unit cell:   57.07  111.11  192.90   90.00   90.07   90.00
>>
>> and
>>
>> Space group: C 1 2 1
>> Average unit cell:  201.10  111.11   57.07   90.00  106.42   90.00
>>
>> Both with Average mosaicity:   0.38 and Rmerge Overall 0.06,
>> but the corresponding MR solutions do not refine, with R-factor stuck to
>> 45-47%.
>>
>> From what I understand, in the I121 SG I have the NC two-fold axis of the
>> dimer at 1/2a and this originates the pseudo centering and the small P3 Cell
>>
>> Largest Patterson peak with length larger than 15 Angstrom:
>>  Frac. coord.  :0.5000.0000.000
>>  Distance to origin:   28.566
>>  Height relative to origin :   53.830 %
>>
>> I'm really not so good with symmetry, so I'll be grateful for any
>> suggestion/help/solution from you out there.
>> Many thanks,
>> Giorgio
>>
>
>

Re: [ccp4bb] doubt regarding setting resolution cutoff

[Christian Roth]

Your plots look strangely different to the old Scala output you posted 
before, but never mind.


Paul is right that a negative intensity is not desired and your crystal 
has some issues with ice.


That one icering around 2.26 must be massive taken into account how 
haywire your curve goes there.


Have you had a look at the images? There should be something visible in 
that area.


Christian


Am 08.08.2017 um 15:17 schrieb Paul Emsley:

On 08/08/2017 15:07, Satvik Kumar wrote:

Dear Prof. Powell and Prof. Dodson,

Thanks for your reply and advise.

As per your suggestion, I have re-scaled the intensities using 
Aimless at 1.861 A.


I observe that the I/sigI has dropped to -0.8 


That's not good.

> and the behaviour of CC_1/2 is still anomalous.

That made me laugh out loud. Perhaps not the best choice of adjective.



Also, when I inspect the Wilson plot (Fig. 1), I observe that the 
curve does not fall smoothly with respect to the reference curve 
(blue). Even with respect to one more Wilson plot from CCP4 website 
(Fig. 2), the curve from my aimless output is different and 
discontinuous.


Icy!

/me wonders if CCP4 are distributing auspex yet...



The second moment of I is constant only up to a resolution of 2.4 Å 
at a value of 3 (Fig. 3). I was not able to get some other plot to 
compare against mine.


Please tell me if I can still go ahead and refine at 1.861 A.


No you can't.

Maybe with some chopping you can rescue some reflections beyond 2.1.

Paul

Re: [ccp4bb] doubt regarding setting resolution cutoff

[Paul Emsley]

On 08/08/2017 15:07, Satvik Kumar wrote:

Dear Prof. Powell and Prof. Dodson,

Thanks for your reply and advise.

As per your suggestion, I have re-scaled the intensities using Aimless at 1.861 
A.

I observe that the I/sigI has dropped to -0.8 


That's not good.

> and the behaviour of CC_1/2 is still anomalous.

That made me laugh out loud. Perhaps not the best choice of adjective.



Also, when I inspect the Wilson plot (Fig. 1), I observe that the curve does not fall smoothly with respect 
to the reference curve (blue). Even with respect to one more Wilson plot from CCP4 website (Fig. 2), the 
curve from my aimless output is different and discontinuous.


Icy!

/me wonders if CCP4 are distributing auspex yet...



The second moment of I is constant only up to a resolution of 2.4 Å at a value of 3 (Fig. 3). I was not able 
to get some other plot to compare against mine.


Please tell me if I can still go ahead and refine at 1.861 A.


No you can't.

Maybe with some chopping you can rescue some reflections beyond 2.1.

Paul

Re: [ccp4bb] pseudo-centering or twinning problem in P3

[Eleanor Dodson]

First of all - check whether your crystal data shows twinning - thee is an
L-test plot which usually gives a clear indication and if you are using
GUI2 the report suggests if this is so.


If you have twinning the most likely SG is P31 (or P32 ) - you cant tell
the difference till the structure is solved.

Check the merging stats in that point group -
Then try MR searches in the two spacegroups.

The C2 and I2 SGs are probably sub-group possibilities if you reindex -
there are common cell dimensions -pointless output tells you how to do that
- but in general one always chooses the highest symmetry that gives
reasonable merging statistics and is sensible.

Eleanor



On 8 August 2017 at 14:15, Giorgio Giardina 
wrote:

> Hello everybody,
>
> I think I have a pseudo-centering problem.
>
> I have a 1.6 ang. dataset of a mutant  protein that is an homodimer in
> solution.
> Data processing gives the following SG:
>
> Space group: P 31 2 1
> Average unit cell:  111.36  111.36   28.56   90.00   90.00  120.00
> Average mosaicity:   0.24
> Rmerge Overall 0.07
>
> However with this SG the unit Cell is too small and monomer doesn't fit
> the in the AU.
>
> I reprocessed the data in other possible SG (including P6) and finally I
> got 2 equivalent SG's in which I can get a correct Molecular Replacement
> solution:
>
> Space group: I 1 2 1
> Average unit cell:   57.07  111.11  192.90   90.00   90.07   90.00
>
> and
>
> Space group: C 1 2 1
> Average unit cell:  201.10  111.11   57.07   90.00  106.42   90.00
>
> Both with Average mosaicity:   0.38 and Rmerge Overall 0.06,
> but the corresponding MR solutions do not refine, with R-factor stuck to
> 45-47%.
>
> From what I understand, in the I121 SG I have the NC two-fold axis of the
> dimer at 1/2a and this originates the pseudo centering and the small P3 Cell
>
> Largest Patterson peak with length larger than 15 Angstrom:
>  Frac. coord.  :0.5000.0000.000
>  Distance to origin:   28.566
>  Height relative to origin :   53.830 %
>
> I'm really not so good with symmetry, so I'll be grateful for any
> suggestion/help/solution from you out there.
> Many thanks,
> Giorgio
>

[ccp4bb] pseudo-centering or twinning problem in P3

[Giorgio Giardina]

Hello everybody,

I think I have a pseudo-centering problem.

I have a 1.6 ang. dataset of a mutant  protein that is an homodimer in solution.
Data processing gives the following SG:

Space group: P 31 2 1
Average unit cell:  111.36  111.36   28.56   90.00   90.00  120.00 
Average mosaicity:   0.24
Rmerge Overall 0.07

However with this SG the unit Cell is too small and monomer doesn't fit the in 
the AU.

I reprocessed the data in other possible SG (including P6) and finally I got 2 
equivalent SG's in which I can get a correct Molecular Replacement solution:

Space group: I 1 2 1 
Average unit cell:   57.07  111.11  192.90   90.00   90.07   90.00 

and

Space group: C 1 2 1
Average unit cell:  201.10  111.11   57.07   90.00  106.42   90.00 

Both with Average mosaicity:   0.38 and Rmerge Overall 0.06,
but the corresponding MR solutions do not refine, with R-factor stuck to 45-47%.

From what I understand, in the I121 SG I have the NC two-fold axis of the dimer 
at 1/2a and this originates the pseudo centering and the small P3 Cell

Largest Patterson peak with length larger than 15 Angstrom:
 Frac. coord.  :0.5000.0000.000
 Distance to origin:   28.566
 Height relative to origin :   53.830 %

I'm really not so good with symmetry, so I'll be grateful for any 
suggestion/help/solution from you out there.
Many thanks,
Giorgio