Re: [ccp4bb] tCNS and space group determination

[Harry Powell]

Hi Marcelo

One thing to try if you have access to a microfocus beamline would be to try to 
collect data from part of the crystal and hope that you hit a single lattice 
with the beam. Sometimes the different lattices are in different parts of the 
physical object in the beam. Recollecting the data on another sample also gives 
you the chance to avoid an icy sample and maybe get a single crystal...

I notice you are using iMosflm to view the image - iMosflm has had a facility 
for processing multiple lattices for many years. It's worth trying, but as Paul 
points out, I wouldn't be optimistic.

Harry
--
Dr Harry Powell
Chairman of European Crystallographic Association SIG9 (Crystallographic 
Computing) 




On 13 Aug 2018, at 08:59, Paul Adams wrote:

> Hi Marcelo,
> 
>  based on this image it looks like you have multiple (two) lattices, which 
> puts spots very close together. This could be a split crystal as Eleanor 
> suggested, or a feature of the way the crystals grow. It seems unlikely that 
> you’d be able to integrate these lattices well, given how close the spots 
> are. You could try data collection with the detector pushed further back to 
> increase spot separation (not an ideal solution). If the multiple lattices is 
> something you see with all crystals then you may want to work on modifying 
> crystallization conditions. 
>  Sometimes things can be very difficult to figure out. The Dicer structure 
> (see Ian MacRae’s paper in Acta D: D63, 993-999, 2007) was a case where the a 
> and b axes of the orthorhombic lattice were very similar in length and the 
> crystals always grew with a pseudo-merohedral twinning that gave rise to 
> nearly, but not quite, overlapped spots from the two lattices. It doesn’t 
> look like you have a case like this, because the intensity statistics don’t 
> suggest twinning. However, some of the tricks that you might use today on 
> such crystals, such as raster scanning with a microbeam, might be helpful.
> 
>  Cheers,
>   Paul
> 
>> On Aug 12, 2018, at 9:57 AM, Randy Read  wrote:
>> 
>> I am sorry. I forgot to attach the image.
>> 
>> Cheers
>> 
>> Marcelo
>> 
>> Em sáb, 11 de ago de 2018 às 18:31, Marcelo Liberato 
>>  escreveu:
>> Dear Eleanor,
>> 
>> Thanks for you answer. 
>> Indeed, there are clear ice rings in the images (example attached). So, I 
>> integrated again (P1, P2 and P222) excluding the resolution ranges 2.28-2.22 
>> and 3.70-3.64. I am attaching the log files from aimless, MR and refmac for 
>> P2 (in two different cells) and P222 data. 
>> I agree that MR seems very good (in all cases), but the final density maps 
>> are always bad. Maybe the data has problems that I am not dealing with. 
>> 
>> Kind regards
>> 
>> Marcelo
>> 
>> Em sáb, 11 de ago de 2018 às 16:04, Eleanor Dodson 
>>  escreveu:
>> This MR looks good to me, but there are serious flaws with the data. Your 
>> secon moment plot from the aimless log has most spectacular spikes which are 
>> always a BAD THING, and the Wilson plot is not very smooth either.. 
>> 
>> As Randy says, try to sort those problems out first.
>> 
>> Then you have this message:
>> 
>> 
>> TRANSLATIONAL NCS:
>> 
>> Translational NCS has been detected at ( 0.000,  0.500,  0.125).
>> A translation of 0.5 along B will generate pseudo-absences along b so you 
>> can be sure whether there is a scre axis or not..
>> 
>> The space group is most likely orthorhombic - these indicators are pretty 
>> convincing for P2/mmm - so I dont know why you have chosen P21 as the 
>> spacegroup? 
>> 
>> 
>> Scores for each symmetry element
>> 
>> Nelmt  Lklhd  Z-ccCCN  RmeasSymmetry & operator (in Lattice 
>> Cell)
>> 
>>  1   0.917   8.18   0.82   61009  0.298 identity
>>  2   0.883   7.85   0.78  100711  0.381 **  2-fold l ( 0 0 1) {-h,-k,l}, 
>> along original k
>>  3   0.921   8.39   0.84   99542  0.355 *** 2-fold k ( 0 1 0) {-h,k,-l}, 
>> along original l
>>  4   0.920   8.26   0.83   99218  0.320 *** 2-fold h ( 1 0 0) {h,-k,-l}, 
>> along original h
>> 
>> So my suggestions:
>> Sort out data problems
>> 
>> Merge as P2/mmm 
>> 
>> Let MR search select the most likely spacegroup of the 8 possible.
>> 
>> You cant even limit the b axis to be a screw axis .
>> 
>> Your refinement behavior looks OK, but the maps will look bad with spurious 
>> reflections in the list..
>> 
>> Eleanor
>> 
>> 
>> 
>> 
>> 
>> On 10 August 2018 at 19:02, Eleanor Dodson  wrote:
>> Actually Marcelo - Refinement to an R of 41% is pretty good for an MR 
>> solution! 
>> 
>> 
>> 
>> On 10 August 2018 at 18:42, Eleanor Dodson  wrote:
>> Can you attach the refinement log?  
>> 
>> Eleanor
>> 
>> On 10 August 2018 at 16:57, Marcelo Liberato  
>> wrote:
>> Dear Randy, 
>> 
>> Thank you very much for answering. I followed your suggestions but, 
>> unfortunately, I couldn't get a reasonable electron density map after MR and 
>> refinement.
>> 
>> 
>> First I would look at the data to see if you have ice rings, because the 
>> peak 

Re: [ccp4bb] tCNS and space group determination

[Eleanor Dodson]

You could try the dials data processing - it tries to address the problems
of multiple lattices..
Eleanor


On 13 August 2018 at 08:59, Paul Adams  wrote:

> Hi Marcelo,
>
>   based on this image it looks like you have multiple (two) lattices,
> which puts spots very close together. This could be a split crystal as
> Eleanor suggested, or a feature of the way the crystals grow. It seems
> unlikely that you’d be able to integrate these lattices well, given how
> close the spots are. You could try data collection with the detector pushed
> further back to increase spot separation (not an ideal solution). If the
> multiple lattices is something you see with all crystals then you may want
> to work on modifying crystallization conditions.
>   Sometimes things can be very difficult to figure out. The Dicer
> structure (see Ian MacRae’s paper in Acta D: D63, 993-999, 2007) was a case
> where the a and b axes of the orthorhombic lattice were very similar in
> length and the crystals always grew with a pseudo-merohedral twinning that
> gave rise to nearly, but not quite, overlapped spots from the two lattices.
> It doesn’t look like you have a case like this, because the intensity
> statistics don’t suggest twinning. However, some of the tricks that you
> might use today on such crystals, such as raster scanning with a microbeam,
> might be helpful.
>
>   Cheers,
> Paul
>
> > On Aug 12, 2018, at 9:57 AM, Randy Read  wrote:
> >
> > I am sorry. I forgot to attach the image.
> >
> > Cheers
> >
> > Marcelo
> >
> > Em sáb, 11 de ago de 2018 às 18:31, Marcelo Liberato <
> marcelovliber...@gmail.com> escreveu:
> > Dear Eleanor,
> >
> > Thanks for you answer.
> > Indeed, there are clear ice rings in the images (example attached). So,
> I integrated again (P1, P2 and P222) excluding the resolution ranges
> 2.28-2.22 and 3.70-3.64. I am attaching the log files from aimless, MR and
> refmac for P2 (in two different cells) and P222 data.
> > I agree that MR seems very good (in all cases), but the final density
> maps are always bad. Maybe the data has problems that I am not dealing
> with.
> >
> > Kind regards
> >
> > Marcelo
> >
> > Em sáb, 11 de ago de 2018 às 16:04, Eleanor Dodson <
> eleanor.dod...@york.ac.uk> escreveu:
> > This MR looks good to me, but there are serious flaws with the data.
> Your secon moment plot from the aimless log has most spectacular spikes
> which are always a BAD THING, and the Wilson plot is not very smooth
> either..
> >
> > As Randy says, try to sort those problems out first.
> >
> > Then you have this message:
> >
> >
> > TRANSLATIONAL NCS:
> >
> > Translational NCS has been detected at ( 0.000,  0.500,  0.125).
> > A translation of 0.5 along B will generate pseudo-absences along b so
> you can be sure whether there is a scre axis or not..
> >
> > The space group is most likely orthorhombic - these indicators are
> pretty convincing for P2/mmm - so I dont know why you have chosen P21 as
> the spacegroup?
> >
> >
> > Scores for each symmetry element
> >
> > Nelmt  Lklhd  Z-ccCCN  RmeasSymmetry & operator (in
> Lattice Cell)
> >
> >   1   0.917   8.18   0.82   61009  0.298 identity
> >   2   0.883   7.85   0.78  100711  0.381 **  2-fold l ( 0 0 1)
> {-h,-k,l}, along original k
> >   3   0.921   8.39   0.84   99542  0.355 *** 2-fold k ( 0 1 0)
> {-h,k,-l}, along original l
> >   4   0.920   8.26   0.83   99218  0.320 *** 2-fold h ( 1 0 0)
> {h,-k,-l}, along original h
> >
> > So my suggestions:
> > Sort out data problems
> >
> > Merge as P2/mmm
> >
> > Let MR search select the most likely spacegroup of the 8 possible.
> >
> > You cant even limit the b axis to be a screw axis .
> >
> > Your refinement behavior looks OK, but the maps will look bad with
> spurious reflections in the list..
> >
> > Eleanor
> >
> >
> >
> >
> >
> > On 10 August 2018 at 19:02, Eleanor Dodson 
> wrote:
> > Actually Marcelo - Refinement to an R of 41% is pretty good for an MR
> solution!
> >
> >
> >
> > On 10 August 2018 at 18:42, Eleanor Dodson 
> wrote:
> > Can you attach the refinement log?
> >
> > Eleanor
> >
> > On 10 August 2018 at 16:57, Marcelo Liberato 
> wrote:
> > Dear Randy,
> >
> > Thank you very much for answering. I followed your suggestions but,
> unfortunately, I couldn't get a reasonable electron density map after MR
> and refinement.
> >
> >
> > First I would look at the data to see if you have ice rings, because the
> peak in mean intensity and second moment of the intensity at about 2.25A
> resolution suggests an ice ring problem.  If so, you should make sure you
> don't contaminate the data with spurious large intensities.
> >
> > Indeed, the data has ice rings. At first, I required imosflm to remove
> ice rings, but it didn't happened. So, I re-processed the data in different
> space groups removing the ice rings.
> >
> > Second, the statistics (e.g. the second moments plot after tNCS
> correction in Phaser) would be consistent with a scenario in which you have
> 

Re: [ccp4bb] tCNS and space group determination

[Paul Adams]

Hi Marcelo,

  based on this image it looks like you have multiple (two) lattices, which 
puts spots very close together. This could be a split crystal as Eleanor 
suggested, or a feature of the way the crystals grow. It seems unlikely that 
you’d be able to integrate these lattices well, given how close the spots are. 
You could try data collection with the detector pushed further back to increase 
spot separation (not an ideal solution). If the multiple lattices is something 
you see with all crystals then you may want to work on modifying 
crystallization conditions. 
  Sometimes things can be very difficult to figure out. The Dicer structure 
(see Ian MacRae’s paper in Acta D: D63, 993-999, 2007) was a case where the a 
and b axes of the orthorhombic lattice were very similar in length and the 
crystals always grew with a pseudo-merohedral twinning that gave rise to 
nearly, but not quite, overlapped spots from the two lattices. It doesn’t look 
like you have a case like this, because the intensity statistics don’t suggest 
twinning. However, some of the tricks that you might use today on such 
crystals, such as raster scanning with a microbeam, might be helpful.

  Cheers,
Paul

> On Aug 12, 2018, at 9:57 AM, Randy Read  wrote:
> 
> I am sorry. I forgot to attach the image.
> 
> Cheers
> 
> Marcelo
> 
> Em sáb, 11 de ago de 2018 às 18:31, Marcelo Liberato 
>  escreveu:
> Dear Eleanor,
> 
> Thanks for you answer. 
> Indeed, there are clear ice rings in the images (example attached). So, I 
> integrated again (P1, P2 and P222) excluding the resolution ranges 2.28-2.22 
> and 3.70-3.64. I am attaching the log files from aimless, MR and refmac for 
> P2 (in two different cells) and P222 data. 
> I agree that MR seems very good (in all cases), but the final density maps 
> are always bad. Maybe the data has problems that I am not dealing with. 
> 
> Kind regards
> 
> Marcelo
> 
> Em sáb, 11 de ago de 2018 às 16:04, Eleanor Dodson 
>  escreveu:
> This MR looks good to me, but there are serious flaws with the data. Your 
> secon moment plot from the aimless log has most spectacular spikes which are 
> always a BAD THING, and the Wilson plot is not very smooth either.. 
> 
> As Randy says, try to sort those problems out first.
> 
> Then you have this message:
> 
> 
> TRANSLATIONAL NCS:
> 
> Translational NCS has been detected at ( 0.000,  0.500,  0.125).
> A translation of 0.5 along B will generate pseudo-absences along b so you can 
> be sure whether there is a scre axis or not..
> 
> The space group is most likely orthorhombic - these indicators are pretty 
> convincing for P2/mmm - so I dont know why you have chosen P21 as the 
> spacegroup? 
> 
> 
> Scores for each symmetry element
> 
> Nelmt  Lklhd  Z-ccCCN  RmeasSymmetry & operator (in Lattice 
> Cell)
> 
>   1   0.917   8.18   0.82   61009  0.298 identity
>   2   0.883   7.85   0.78  100711  0.381 **  2-fold l ( 0 0 1) {-h,-k,l}, 
> along original k
>   3   0.921   8.39   0.84   99542  0.355 *** 2-fold k ( 0 1 0) {-h,k,-l}, 
> along original l
>   4   0.920   8.26   0.83   99218  0.320 *** 2-fold h ( 1 0 0) {h,-k,-l}, 
> along original h
> 
> So my suggestions:
> Sort out data problems
> 
> Merge as P2/mmm 
> 
> Let MR search select the most likely spacegroup of the 8 possible.
> 
> You cant even limit the b axis to be a screw axis .
> 
> Your refinement behavior looks OK, but the maps will look bad with spurious 
> reflections in the list..
> 
> Eleanor
> 
> 
> 
> 
> 
> On 10 August 2018 at 19:02, Eleanor Dodson  wrote:
> Actually Marcelo - Refinement to an R of 41% is pretty good for an MR 
> solution! 
> 
> 
> 
> On 10 August 2018 at 18:42, Eleanor Dodson  wrote:
> Can you attach the refinement log?  
> 
> Eleanor
> 
> On 10 August 2018 at 16:57, Marcelo Liberato  
> wrote:
> Dear Randy, 
> 
> Thank you very much for answering. I followed your suggestions but, 
> unfortunately, I couldn't get a reasonable electron density map after MR and 
> refinement.
> 
> 
> First I would look at the data to see if you have ice rings, because the peak 
> in mean intensity and second moment of the intensity at about 2.25A 
> resolution suggests an ice ring problem.  If so, you should make sure you 
> don't contaminate the data with spurious large intensities.
> 
> Indeed, the data has ice rings. At first, I required imosflm to remove ice 
> rings, but it didn't happened. So, I re-processed the data in different space 
> groups removing the ice rings.  
> 
> Second, the statistics (e.g. the second moments plot after tNCS correction in 
> Phaser) would be consistent with a scenario in which you have pseudosymmetry 
> along with a twin operator that parallels the pseudosymmetry.  If that's 
> true, it's hard to be sure of the symmetry.  For instance, if the structure 
> really is monoclinic, can you be sure you chose the correct axis to be the 
> 2-fold?
>  
> I am not sure. However, I tried two possible axis to be the 2-fold and none 
> 

Re: [ccp4bb] tCNS and space group determination

[Randy Read]

Hi,

Looking at the pointless logfile for P222, there’s excellent evidence for a 
2(1) screw along the shortest and longest cell edges as Eleanor says, which 
would make the space group P 21 2 21 for the data set used in the Phaser run 
with the data merged in P222.  The merging statistics are equally good for all 
3 2-folds and for the identity, which implies, as Eleanor says, that the data 
most likely should be merged as orthorhombic.  If the data were twinned that 
would explain merging in higher symmetry even if the true space group were some 
version of P2 or P21, but the twinning statistics look pretty close to what one 
would expect for an untwinned crystal.

Maybe you’ve run Phaser in all 8 possible orthorhombic space groups in some 
other job, but in the job that the file name implies was run in P212121, it was 
actually only run in P222, i.e. the point group in which the data were merged.  
So this job has missed all the more likely space groups.

What Phaser flags as an outlier rejection changed a couple of years ago, though 
we still haven’t published this.  For a long time, Phaser has rejected 
improbably large structure factors, i.e. ones that would be expected to occur 
less than one time in a million according to the Wilson distribution.  For 
about 2 years now, it has also been ignoring reflections flagged as containing 
very little information about the true intensity, i.e. ones in which the 
standard deviation of the intensity (SIGI) is large compared to the Wilson 
expected intensity.  When a crystal has very high anisotropy (as in this case, 
where the anisotropic delta B, or the difference between the weakest and 
strongest directions, is nearly 60 A^2, many of the reflections in the weak 
directions in reciprocal space will contain so little information that they can 
be ignored in the calculation (because they would contribute almost nothing 
apart from raising the CPU time!).  The presence of tNCS introduces more 
reflections that are systematically very weak, as well.  I should probably 
change the log file output to separate the counts for the rejections vs the 
ones being ignored.  There are data sets where nearly half of the reflections 
end up being ignored, so I wouldn’t worry about that for this data set.

Best wishes,

Randy Read

-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical ResearchTel: +44 1223 336500
Wellcome Trust/MRC Building Fax: +44 1223 336827
Hills RoadE-mail: 
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   
www-structmed.cimr.cam.ac.uk

> On 11 Aug 2018, at 20:36, Eleanor Dodson 
> <176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Marcelo - there is something very wrong with the data. You dont to reprocess 
> in other space groups - the P2/mmm symmetry looks convincing but PHASER says 
> there are 3500 rejections! That is an awful lot - 10 is a more normal value. 
> I cant see the image clearly but something is causing problems. Split 
> crystal? How are you processing it?
> 
> The Moments go crazy at high resolution. Maybe try redoing the MR & 
> refinement  all at 3A? 
>  
> 
> The MR rotation function solution is wonderful, but you need to conside 
> spacegroups P 21 2 21. and P 21 21 21 .
> The translation vector of 0 0.5 0.13 would produce absences along the b axis. 
> The screw axes along a and c look safe. 
> 
> 
> 
> On 11 August 2018 at 19:36, Marcelo Liberato  
> wrote:
> I am sorry. I forgot to attach the image.
> 
> Cheers
> 
> Marcelo
> 
> Em sáb, 11 de ago de 2018 às 18:31, Marcelo Liberato 
>  escreveu:
> Dear Eleanor,
> 
> Thanks for you answer. 
> Indeed, there are clear ice rings in the images (example attached). So, I 
> integrated again (P1, P2 and P222) excluding the resolution ranges 2.28-2.22 
> and 3.70-3.64. I am attaching the log files from aimless, MR and refmac for 
> P2 (in two different cells) and P222 data. 
> I agree that MR seems very good (in all cases), but the final density maps 
> are always bad. Maybe the data has problems that I am not dealing with. 
> 
> Kind regards
> 
> Marcelo
> 
> Em sáb, 11 de ago de 2018 às 16:04, Eleanor Dodson 
>  escreveu:
> This MR looks good to me, but there are serious flaws with the data. Your 
> secon moment plot from the aimless log has most spectacular spikes which are 
> always a BAD THING, and the Wilson plot is not very smooth either.. 
> 
> As Randy says, try to sort those problems out first.
> 
> Then you have this message:
> 
> 
> TRANSLATIONAL NCS:
> 
> Translational NCS has been detected at ( 0.000,  0.500,  0.125).
> A translation of 0.5 along B will generate pseudo-absences along b so you can 
> be sure whether there is a scre axis or not..
> 
> The space group is most likely orthorhombic - these indicators are pretty 
> convincing for P2/mmm - so I dont know why you have chosen P21 as the 
> spacegroup? 

Re: [ccp4bb] tCNS and space group determination

[Eleanor Dodson]

Marcelo - there is something very wrong with the data. You dont to
reprocess in other space groups - the P2/mmm symmetry looks convincing but
PHASER says there are 3500 rejections! That is an awful lot - 10 is a more
normal value.
I cant see the image clearly but something is causing problems. Split
crystal? How are you processing it?

The Moments go crazy at high resolution. Maybe try redoing the MR &
refinement  all at 3A?


The MR rotation function solution is wonderful, but you need to conside
spacegroups P 21 2 21. and P 21 21 21 .
The translation vector of 0 0.5 0.13 would produce absences along the b
axis.
The screw axes along a and c look safe.



On 11 August 2018 at 19:36, Marcelo Liberato 
wrote:

> I am sorry. I forgot to attach the image.
>
> Cheers
>
> Marcelo
>
> Em sáb, 11 de ago de 2018 às 18:31, Marcelo Liberato <
> marcelovliber...@gmail.com> escreveu:
>
>> Dear Eleanor,
>>
>> Thanks for you answer.
>> Indeed, there are clear ice rings in the images (example attached). So, I
>> integrated again (P1, P2 and P222) excluding the resolution ranges 2.28-2.22
>> and 3.70-3.64. I am attaching the log files from aimless, MR and refmac
>> for P2 (in two different cells) and P222 data.
>> I agree that MR seems very good (in all cases), but the final density
>> maps are always bad. Maybe the data has problems that I am not dealing
>> with.
>>
>> Kind regards
>>
>> Marcelo
>>
>> Em sáb, 11 de ago de 2018 às 16:04, Eleanor Dodson <
>> eleanor.dod...@york.ac.uk> escreveu:
>>
>>> This MR looks good to me, but there are serious flaws with the data.
>>> Your secon moment plot from the aimless log has most spectacular spikes
>>> which are always a BAD THING, and the Wilson plot is not very smooth
>>> either..
>>>
>>> As Randy says, try to sort those problems out first.
>>>
>>> Then you have this message:
>>>
>>>
>>> TRANSLATIONAL NCS:
>>>
>>> Translational NCS has been detected at ( 0.000,  0.500,  0.125).
>>> A translation of 0.5 along B will generate pseudo-absences along b so
>>> you can be sure whether there is a scre axis or not..
>>>
>>> The space group is most likely orthorhombic - these indicators are
>>> pretty convincing for P2/mmm - so I dont know why you have chosen P21 as
>>> the spacegroup?
>>>
>>>
>>> Scores for each symmetry element
>>>
>>> Nelmt  Lklhd  Z-ccCCN  RmeasSymmetry & operator (in
>>> Lattice Cell)
>>>
>>>   1   *0.917 *  8.18   0.82   61009  0.298 identity
>>>   2  * 0.883*   7.85   0.78  100711  0.381 **  2-fold l ( 0 0 1)
>>> {-h,-k,l}, along original k
>>>   3   *0.921*   8.39   0.84   99542  0.355 *** 2-fold k ( 0 1 0)
>>> {-h,k,-l}, along original l
>>>   4   *0.920*   8.26   0.83   99218  0.320 *** 2-fold h ( 1 0 0)
>>> {h,-k,-l}, along original h
>>>
>>> So my suggestions:
>>> Sort out data problems
>>>
>>> Merge as P2/mmm
>>>
>>> Let MR search select the most likely spacegroup of the 8 possible.
>>>
>>> You cant even limit the b axis to be a screw axis .
>>>
>>> Your refinement behavior looks OK, but the maps will look bad with
>>> spurious reflections in the list..
>>>
>>> Eleanor
>>>
>>>
>>>
>>>
>>>
>>> On 10 August 2018 at 19:02, Eleanor Dodson 
>>> wrote:
>>>
 Actually Marcelo - Refinement to an R of 41% is pretty good for an MR
 solution!



 On 10 August 2018 at 18:42, Eleanor Dodson 
 wrote:

> Can you attach the refinement log?
>
> Eleanor
>
> On 10 August 2018 at 16:57, Marcelo Liberato <
> marcelovliber...@gmail.com> wrote:
>
>> Dear Randy,
>>
>> Thank you very much for answering. I followed your suggestions but,
>> unfortunately, I couldn't get a reasonable electron density map after MR
>> and refinement.
>>
>>
>> First I would look at the data to see if you have ice rings, because
>>> the peak in mean intensity and second moment of the intensity at about
>>> 2.25A resolution suggests an ice ring problem.  If so, you should make 
>>> sure
>>> you don't contaminate the data with spurious large intensities.
>>>
>>> Indeed, the data has ice rings. At first, I required imosflm to
>> remove ice rings, but it didn't happened. So, I re-processed the data in
>> different space groups removing the ice rings.
>>
>> Second, the statistics (e.g. the second moments plot after tNCS
>>> correction in Phaser) would be consistent with a scenario in which you 
>>> have
>>> pseudosymmetry along with a twin operator that parallels the
>>> pseudosymmetry.  If that's true, it's hard to be sure of the symmetry.  
>>> For
>>> instance, if the structure really is monoclinic, can you be sure you 
>>> chose
>>> the correct axis to be the 2-fold?
>>>
>>
>> I am not sure. However, I tried two possible axis to be the 2-fold
>> and none of them gave me reasonable maps after MR and refinement.
>>
>>
>>>
>>> Since you have a good model that gives clear MR solutions even in

Re: [ccp4bb] tCNS and space group determination

[Eleanor Dodson]

This MR looks good to me, but there are serious flaws with the data. Your
secon moment plot from the aimless log has most spectacular spikes which
are always a BAD THING, and the Wilson plot is not very smooth either..

As Randy says, try to sort those problems out first.

Then you have this message:


TRANSLATIONAL NCS:

Translational NCS has been detected at ( 0.000,  0.500,  0.125).
A translation of 0.5 along B will generate pseudo-absences along b so you
can be sure whether there is a scre axis or not..

The space group is most likely orthorhombic - these indicators are pretty
convincing for P2/mmm - so I dont know why you have chosen P21 as the
spacegroup?


Scores for each symmetry element

Nelmt  Lklhd  Z-ccCCN  RmeasSymmetry & operator (in Lattice
Cell)

  1   *0.917 *  8.18   0.82   61009  0.298 identity
  2  * 0.883*   7.85   0.78  100711  0.381 **  2-fold l ( 0 0 1) {-h,-k,l},
along original k
  3   *0.921*   8.39   0.84   99542  0.355 *** 2-fold k ( 0 1 0) {-h,k,-l},
along original l
  4   *0.920*   8.26   0.83   99218  0.320 *** 2-fold h ( 1 0 0) {h,-k,-l},
along original h

So my suggestions:
Sort out data problems

Merge as P2/mmm

Let MR search select the most likely spacegroup of the 8 possible.

You cant even limit the b axis to be a screw axis .

Your refinement behavior looks OK, but the maps will look bad with spurious
reflections in the list..

Eleanor





On 10 August 2018 at 19:02, Eleanor Dodson 
wrote:

> Actually Marcelo - Refinement to an R of 41% is pretty good for an MR
> solution!
>
>
>
> On 10 August 2018 at 18:42, Eleanor Dodson 
> wrote:
>
>> Can you attach the refinement log?
>>
>> Eleanor
>>
>> On 10 August 2018 at 16:57, Marcelo Liberato 
>> wrote:
>>
>>> Dear Randy,
>>>
>>> Thank you very much for answering. I followed your suggestions but,
>>> unfortunately, I couldn't get a reasonable electron density map after MR
>>> and refinement.
>>>
>>>
>>> First I would look at the data to see if you have ice rings, because the
 peak in mean intensity and second moment of the intensity at about 2.25A
 resolution suggests an ice ring problem.  If so, you should make sure you
 don't contaminate the data with spurious large intensities.

 Indeed, the data has ice rings. At first, I required imosflm to remove
>>> ice rings, but it didn't happened. So, I re-processed the data in different
>>> space groups removing the ice rings.
>>>
>>> Second, the statistics (e.g. the second moments plot after tNCS
 correction in Phaser) would be consistent with a scenario in which you have
 pseudosymmetry along with a twin operator that parallels the
 pseudosymmetry.  If that's true, it's hard to be sure of the symmetry.  For
 instance, if the structure really is monoclinic, can you be sure you chose
 the correct axis to be the 2-fold?

>>>
>>> I am not sure. However, I tried two possible axis to be the 2-fold and
>>> none of them gave me reasonable maps after MR and refinement.
>>>
>>>

 Since you have a good model that gives clear MR solutions even in P21,
 you can probably process the data in P1 and solve it with 8 copies in the
 unit cell.  Then you can look at the symmetry of the MR solution (e.g. in
 Zanuda) and see whether it obeys any higher symmetry than P1.

>>>
>>> I processed data in P1. After MR (with 8 copies in the ASU), it
>>> resulted in TFZ=11.6 and LLG=1434. But the map is still bad and high
>>> Rwork and Rfree.
>>> According to Zanuda, the data should be P21:
>>>
>>> ^
>>>
>>>| >>   4   | P 1 21 1   | 68.6868  |  0.6289  |  0.5487  |  0.5523  |
>>>
>>>-
>>>
>>>|  1   | P 1| 69.4151  |  0.6171  |  0.5471  |  0.5559  |
>>>
>>>|  4   | P 1 21 1   | 69.3810  |--|  0.5482  |  0.5442  |
>>>
>>>| 11   | P 21 21 21 | 52.0271  |--|  0.6107  |  0.6178  |
>>>
>>>-
>>>
>>>| <<   4   | P 1 21 1   | 69.3810  |--|  0.5482  |  0.5442  |
>>>
>>>-
>>>
>>> I processed in P21 using two different unit cells, and MR resulted in
>>> TFZ=20.8 and LLG=511, and TFZ=56 and LLG=2867. However, again, no good
>>> maps and statistics.
>>>
>>> Best regards
>>>
>>> Marcelo Liberato
>>>
>>> --
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>>
>>
>>
>



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Re: [ccp4bb] tCNS and space group determination

[Eleanor Dodson]

Actually Marcelo - Refinement to an R of 41% is pretty good for an MR
solution!



On 10 August 2018 at 18:42, Eleanor Dodson 
wrote:

> Can you attach the refinement log?
>
> Eleanor
>
> On 10 August 2018 at 16:57, Marcelo Liberato 
> wrote:
>
>> Dear Randy,
>>
>> Thank you very much for answering. I followed your suggestions but,
>> unfortunately, I couldn't get a reasonable electron density map after MR
>> and refinement.
>>
>>
>> First I would look at the data to see if you have ice rings, because the
>>> peak in mean intensity and second moment of the intensity at about 2.25A
>>> resolution suggests an ice ring problem.  If so, you should make sure you
>>> don't contaminate the data with spurious large intensities.
>>>
>>> Indeed, the data has ice rings. At first, I required imosflm to remove
>> ice rings, but it didn't happened. So, I re-processed the data in different
>> space groups removing the ice rings.
>>
>> Second, the statistics (e.g. the second moments plot after tNCS
>>> correction in Phaser) would be consistent with a scenario in which you have
>>> pseudosymmetry along with a twin operator that parallels the
>>> pseudosymmetry.  If that's true, it's hard to be sure of the symmetry.  For
>>> instance, if the structure really is monoclinic, can you be sure you chose
>>> the correct axis to be the 2-fold?
>>>
>>
>> I am not sure. However, I tried two possible axis to be the 2-fold and
>> none of them gave me reasonable maps after MR and refinement.
>>
>>
>>>
>>> Since you have a good model that gives clear MR solutions even in P21,
>>> you can probably process the data in P1 and solve it with 8 copies in the
>>> unit cell.  Then you can look at the symmetry of the MR solution (e.g. in
>>> Zanuda) and see whether it obeys any higher symmetry than P1.
>>>
>>
>> I processed data in P1. After MR (with 8 copies in the ASU), it resulted
>> in TFZ=11.6 and LLG=1434. But the map is still bad and high Rwork and
>> Rfree.
>> According to Zanuda, the data should be P21:
>>
>> ^
>>
>>| >>   4   | P 1 21 1   | 68.6868  |  0.6289  |  0.5487  |  0.5523  |
>>
>>-
>>
>>|  1   | P 1| 69.4151  |  0.6171  |  0.5471  |  0.5559  |
>>
>>|  4   | P 1 21 1   | 69.3810  |--|  0.5482  |  0.5442  |
>>
>>| 11   | P 21 21 21 | 52.0271  |--|  0.6107  |  0.6178  |
>>
>>-
>>
>>| <<   4   | P 1 21 1   | 69.3810  |--|  0.5482  |  0.5442  |
>>
>>-
>>
>> I processed in P21 using two different unit cells, and MR resulted in 
>> TFZ=20.8
>> and LLG=511, and TFZ=56 and LLG=2867. However, again, no good maps and
>> statistics.
>>
>> Best regards
>>
>> Marcelo Liberato
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>
>



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Re: [ccp4bb] tCNS and space group determination

[Eleanor Dodson]

Can you attach the refinement log?

Eleanor

On 10 August 2018 at 16:57, Marcelo Liberato 
wrote:

> Dear Randy,
>
> Thank you very much for answering. I followed your suggestions but,
> unfortunately, I couldn't get a reasonable electron density map after MR
> and refinement.
>
>
> First I would look at the data to see if you have ice rings, because the
>> peak in mean intensity and second moment of the intensity at about 2.25A
>> resolution suggests an ice ring problem.  If so, you should make sure you
>> don't contaminate the data with spurious large intensities.
>>
>> Indeed, the data has ice rings. At first, I required imosflm to remove
> ice rings, but it didn't happened. So, I re-processed the data in different
> space groups removing the ice rings.
>
> Second, the statistics (e.g. the second moments plot after tNCS correction
>> in Phaser) would be consistent with a scenario in which you have
>> pseudosymmetry along with a twin operator that parallels the
>> pseudosymmetry.  If that's true, it's hard to be sure of the symmetry.  For
>> instance, if the structure really is monoclinic, can you be sure you chose
>> the correct axis to be the 2-fold?
>>
>
> I am not sure. However, I tried two possible axis to be the 2-fold and
> none of them gave me reasonable maps after MR and refinement.
>
>
>>
>> Since you have a good model that gives clear MR solutions even in P21,
>> you can probably process the data in P1 and solve it with 8 copies in the
>> unit cell.  Then you can look at the symmetry of the MR solution (e.g. in
>> Zanuda) and see whether it obeys any higher symmetry than P1.
>>
>
> I processed data in P1. After MR (with 8 copies in the ASU), it resulted
> in TFZ=11.6 and LLG=1434. But the map is still bad and high Rwork and
> Rfree.
> According to Zanuda, the data should be P21:
>
> ^
>
>| >>   4   | P 1 21 1   | 68.6868  |  0.6289  |  0.5487  |  0.5523  |
>
>-
>
>|  1   | P 1| 69.4151  |  0.6171  |  0.5471  |  0.5559  |
>
>|  4   | P 1 21 1   | 69.3810  |--|  0.5482  |  0.5442  |
>
>| 11   | P 21 21 21 | 52.0271  |--|  0.6107  |  0.6178  |
>
>-
>
>| <<   4   | P 1 21 1   | 69.3810  |--|  0.5482  |  0.5442  |
>
>-
>
> I processed in P21 using two different unit cells, and MR resulted in TFZ=20.8
> and LLG=511, and TFZ=56 and LLG=2867. However, again, no good maps and
> statistics.
>
> Best regards
>
> Marcelo Liberato
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] tCNS and space group determination

[Marcelo Liberato]

Dear Randy,

Thank you very much for answering. I followed your suggestions but,
unfortunately, I couldn't get a reasonable electron density map after MR
and refinement.


First I would look at the data to see if you have ice rings, because the
> peak in mean intensity and second moment of the intensity at about 2.25A
> resolution suggests an ice ring problem.  If so, you should make sure you
> don't contaminate the data with spurious large intensities.
>
> Indeed, the data has ice rings. At first, I required imosflm to remove
ice rings, but it didn't happened. So, I re-processed the data in different
space groups removing the ice rings.

Second, the statistics (e.g. the second moments plot after tNCS correction
> in Phaser) would be consistent with a scenario in which you have
> pseudosymmetry along with a twin operator that parallels the
> pseudosymmetry.  If that's true, it's hard to be sure of the symmetry.  For
> instance, if the structure really is monoclinic, can you be sure you chose
> the correct axis to be the 2-fold?
>

I am not sure. However, I tried two possible axis to be the 2-fold and none
of them gave me reasonable maps after MR and refinement.


>
> Since you have a good model that gives clear MR solutions even in P21, you
> can probably process the data in P1 and solve it with 8 copies in the unit
> cell.  Then you can look at the symmetry of the MR solution (e.g. in
> Zanuda) and see whether it obeys any higher symmetry than P1.
>

I processed data in P1. After MR (with 8 copies in the ASU), it resulted in
TFZ=11.6 and LLG=1434. But the map is still bad and high Rwork and Rfree.
According to Zanuda, the data should be P21:

^

   | >>   4   | P 1 21 1   | 68.6868  |  0.6289  |  0.5487  |  0.5523  |

   -

   |  1   | P 1| 69.4151  |  0.6171  |  0.5471  |  0.5559  |

   |  4   | P 1 21 1   | 69.3810  |--|  0.5482  |  0.5442  |

   | 11   | P 21 21 21 | 52.0271  |--|  0.6107  |  0.6178  |

   -

   | <<   4   | P 1 21 1   | 69.3810  |--|  0.5482  |  0.5442  |

   -

I processed in P21 using two different unit cells, and MR resulted in TFZ=20.8
and LLG=511, and TFZ=56 and LLG=2867. However, again, no good maps and
statistics.

Best regards

Marcelo Liberato



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Re: [ccp4bb] tCNS and space group determination

[Randy Read]

Dear Marcelo,

First I would look at the data to see if you have ice rings, because the peak 
in mean intensity and second moment of the intensity at about 2.25A resolution 
suggests an ice ring problem.  If so, you should make sure you don't 
contaminate the data with spurious large intensities.

Second, the statistics (e.g. the second moments plot after tNCS correction in 
Phaser) would be consistent with a scenario in which you have pseudosymmetry 
along with a twin operator that parallels the pseudosymmetry.  If that's true, 
it's hard to be sure of the symmetry.  For instance, if the structure really is 
monoclinic, can you be sure you chose the correct axis to be the 2-fold?

Since you have a good model that gives clear MR solutions even in P21, you can 
probably process the data in P1 and solve it with 8 copies in the unit cell.  
Then you can look at the symmetry of the MR solution (e.g. in Zanuda) and see 
whether it obeys any higher symmetry than P1.

Good luck!

Randy Read

> On 7 Aug 2018, at 10:35, Marcelo Liberato  wrote:
> 
> Dear CCP4 members, 
> 
> I need your help to figure out what is going on with my data. 
> I've integrated my data set in space groups P1, P2 and P222. All with cell 
> parameters: 45 89 149 90 90 90.
> 
> Aimless always indicates P212121 as the correct space group and the 
> resolution is about 2.0 A.
> 
> I have tried MR (Phaser) with all these space groups (and the alternative 
> ones) using a trimmed model (I've cut some loops) with 37 % identity (besides 
> the low identity, this enzyme belongs to a protein family with high 
> structural similarity). In most cases MR resulted in TFZ between 12-19 and 
> LLG 200-400. 
> However, refinement with refmac (with and without twin) generally results in 
> Rwork/Rfree ~ 0.4400/0.4800. 
> The best scenario was obtained when data was integrated in P21. MR resulted 
> in TFZ=19 and LLG=450, and refmac (with twin) resulted in Rwork/Rfree = 
> 0.4129/0.4517. This data was used in Autobuild (Phenix), twin law h,-k,-l,  
> resulting in Rwork/Rfree = 0.3525/0.4040. The final model seems to fit the 
> electron density, but the map is not very good and there is a lot of bias. 
> 
> All data generated by aimless and MR in the different space groups were 
> analyzed by Xtriage. In all cases, "Translational pseudo-symmetry is very 
> likely present in these data" with an off-origin peak (38 % of origin peak) 
> at fractional coordinates (0.0, 0.5, 0.13). 
> 
> Now, I am stuck and I have no idea how to solve this problem. Could you 
> please help me with this?
> 
> The log files of aimless, MR, refmac and Xtriage of the "best scenario" are 
> attached. 
> 
> Many thanks
> 
> Marcelo Liberato
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> <49_refmac5.log><46_phaser_MR.log><45_aimless.log>

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk




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