Re: [ccp4bb] Translational NCS (tNCS)

[Eleanor Dodson]

WELL - with such high LLG and translation NCSthe high R factors are a bit
surprising..
The Patterson info does not give the relative height of the origin peak to
the off origin one..
Questions which I would check.
Look at the data processing carefully. Are there bad batches? Could the
space group be wrong? Is the Wilson plot linear? Any twinning? etc..

How many molecules do you expect in the asymmetric unit?

And so on. I can look at your log files if you like and try to see if they
help.
Eleanor

On Tue, 27 Jun 2023 at 18:03, Muhammad Bashir Khan  wrote:

> Hello All;
>
> I have x-rays data of about 100 kDa protein at 3.3A. It gives MR
> statistics as LLG 150 and TFZ 7 but when I switched off the tNCS I got an
> LLG around 2500 and TFZ 45, but the R and Rfree are stuck at 39 and 43. The
> space group is 4. Any suggestion in this regard will be highly appreciated.
> Patterson's analysis is attached.
>
> Thank you all
>
> Bashir
>
> --
> --
> Muhammad Bashir Khan, Ph.D.
> Research Associate
> Department of Biochemistry
> Medical Science Bldg.
> Lab 3-27
> University of Alberta
> Edmonton AB, T6G 2H7
>
> Phone: 780-492-4577-
> e-mail: m...@ualberta.ca 
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Translational NCS (tNCS)

[Esko Oksanen]

Hi Bashir,

It does look like you have translational pseudo-symmetry. If the translation 
vector happens to be body-centering, it could affect your intensity statistics 
so that the R-values would never go down as half of your reflections follow the 
distribution for centric reflections. I had this a long time ago; you might 
want to check https://doi.org/10.1107/S0907444906031519.

HTH,
Esko

Sent from my iPhone

On 27 Jun 2023, at 19:03, Muhammad Bashir Khan  wrote:


Hello All;

I have x-rays data of about 100 kDa protein at 3.3A. It gives MR statistics as 
LLG 150 and TFZ 7 but when I switched off the tNCS I got an LLG around 2500 and 
TFZ 45, but the R and Rfree are stuck at 39 and 43. The space group is 4. Any 
suggestion in this regard will be highly appreciated. Patterson's analysis is 
attached.

Thank you all

Bashir

--
--
Muhammad Bashir Khan, Ph.D.
Research Associate
Department of Biochemistry
Medical Science Bldg.
Lab 3-27
University of Alberta
Edmonton AB, T6G 2H7

Phone: 780-492-4577-
e-mail: m...@ualberta.ca



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Re: [ccp4bb] translational NCS & twinning

[Donghyuk Shin]

Dear Eleanor,

Many thanks for you comments.

I have run aimless/pointless with those data sets having unit cell (134/67, 
134/67, 183, 90, 90 120) previously integrated with P31 2 1.
Previously, I forced aimless to not determine laue group, to keep the original 
SG, and now I let aimless determine the SG.

Both aimless and pointless re-indexed both data sets to the P63 2 2 for both 
data sets.
Based on the matthews analysis, it seems impossible to put molecule in to small 
cell (67. 67, 183, 90, 90, 120), and truncate analysis for this large cell 
indicates both tNCS and twinning. I am confused..how to interpret my data sets. 
Does it have both tNCS and twinning? 

For curiosity, I have ran the Phaser by turning on/off the tNCS with larger 
cell (134, 134, 180, 90, 90, 120), and only the phaser 'without tNCS' gave me 
the solution, but still it did not give me the 2 molecules/ASU which should be, 
and just 1mol/ASU.
Again for curiosity, I ran Refmac but results were like below.
-> Refmac without twin: 0.51/0.56 (work/free)
-> Refmac with twin: 0.52/0.59

I am also attached the log file of pointless for both cells.

Going back to the previous post, I am very open to accept that my C2 refinement 
is wrong, happy to learn. But, based on L-test, H-test and dropping R-values 
and model with density, I guess this is quite convincing but as you commented 
maybe I am misleading. Please let me know if you have more comments.


Best wishes,
Donghyuk





To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
#CCP4I VERSION CCP4Interface 7.0.051
#CCP4I SCRIPT LOG pointless
#CCP4I DATE 15 Jan 2019  13:42:04
#CCP4I USER user
#CCP4I PROJECT donghyuk_d011_x024
#CCP4I JOB_ID 135
#CCP4I SCRATCH /tmp/user
#CCP4I HOSTNAME localhost.localdomain
#CCP4I PID 30946

 
 ###
 ###
 ###
 ### CCP4 7.0.051: POINTLESS version 1.11.8 : 19/12/17##
 ###
 User: user  Run date: 15/ 1/2019 Run time: 13:42:04 


 Please reference: Collaborative Computational Project, Number 4. 2011.
 "Overview of the CCP4 suite and current developments". Acta Cryst. D67, 235-242.
 as well as any specific reference in the program write-up.

 Input command lines 

XDSIN /home/user/Donghyuk/d011_x024/process_donghyuk/xds_016/XDS_ASCII.HKL
HKLOUT /home/user/Donghyuk/d011_x024/ccp4/XDS_016_pointless.mtz
## This script run with the command   ##
# /home/user/Downloads/destination/ccp4-7.0/bin/pointless


 End of input

Release Date: 19th December 2017


**
**
* POINTLESS  *
*   1.11.8   *
**
*   Determine Laue group from unmerged intensities   *
* Phil Evans MRC LMB, Cambridge  *
* Uses cctbx routines by Ralf Grosse-Kunstleve et al.*
**
**


Reading XDS ascii file from file /home/user/Donghyuk/d011_x024/process_donghyuk/xds_016/XDS_ASCII.HKL

Header lines:

!FORMAT=XDS_ASCIIMERGE=FALSEFRIEDEL'S_LAW=FALSE
!OUTPUT_FILE=XDS_ASCII.HKLDATE=11-Jan-2019
!Generated by CORRECT   (VERSION Jan 26, 2018  BUILT=20180808)
!PROFILE_FITTING= TRUE 
!NAME_TEMPLATE_OF_DATA_FRAMES=/home/user/Donghyuk/d011_x024/x024_C11_1_??.h5 GENERIC
!DATA_RANGE=   1 900
!ROTATION_AXIS=  0.99  0.001086 -0.000195
!OSCILLATION_RANGE=  0.15
!STARTING_ANGLE= 0.000
!STARTING_FRAME=   1
!INCLUDE_RESOLUTION_RANGE=50.000 1.582
!SPACE_GROUP_NUMBER=  152
!UNIT_CELL_CONSTANTS=   134.222   134.222   182.665  90.000  90.000 120.000
!UNIT_CELL_A-AXIS=-9.656   -13.195   133.223
!UNIT_CELL_B-AXIS=36.958   117.512   -53.297
!UNIT_CELL_C-AXIS=  -175.05651.620-7.575
!REFLECTING_RANGE_E.S.D.= 0.163
!BEAM_DIVERGENCE_E.S.D.= 0.029
!X-RAY_WAVELENGTH=  0.87
!INCIDENT_BEAM_DIRECTION= -0.002009 -0.001292  1.10
!FRACTION_OF_POLARIZATION=   0.990
!POLARIZATION_PLANE_NORMAL=  0.00  1.00  0.00
!AIR=  0.000339
!SILICON=  3.942720
!SENSOR_THICKNESS=  0.45
!DETECTOR=EIGER 
!OVERLOAD=   300
!NX=  4150  NY=  4371QX=  0.075000  QY=  0.075000
!ORGX=   2066.92  ORGY=   2186.64
!DETECTOR_DISTANCE=   260.894
!DIRECTION_OF_DETECTOR_X-AXIS=   1.0   0.0   0.0
!DIRECTION_OF_DETECTOR_Y-AXIS=   

Re: [ccp4bb] translational NCS & twinning

[Eleanor Dodson]

Donghyuk - testing your "twinning" in a lower symmetry space group can be
misleading.
Many checks look to see if twin related reflections are similar.
But the twin laws are often the same as the symmetry operators, so if you
have ignored the true symmetry, you will wrongly assume you have twinning.

In this case I would do the following.
Look at the small trigonal cell in pointless/aimless etc.  (134.223/2
134.223/2 182.666, 90, 90, 120)
(You can just input the data integrated in C2 I think and pointless will
check alternate cells.
Your C2  data with these cell dimensions can be reindexed into a trigonal
cell
67.2  67.2 182.9  90.0  90.0 120.0 )

Then pointless will check the likely pointgroup symmetrys -  P3 ?  P3/mmm
?  P6?  etc and give you a score for each symmetry operator.

And since you now have no non-cryst translation you can believe the twin
tests much more.

OK?
Eleanor









On Tue, 15 Jan 2019 at 10:57, Donghyuk Shin  wrote:

> Dear all,
>
> Thank you very much for all of your constructive comments.
> Based on all of your comments, I have done several treatment on my data
> sets, and here are some questions related with both your comments and my
> results.
>
> First of all, I would like to make a short note summarizing all of your
> comments for the people who will have similar problem.
>
> 1) Twinning and tNCS has opposite effects to each other, and one should
> carefully analyze the data if one of them present.
> 2) Simple tNCS can be automatically analyzed and corrected during Phaser,
> but if it does not work, you may try to lower symmetry, decrease the size
> of the Uni cell, or turning off the tNCS option during phaser.
> 3) If one think there is twinning in the crystal, to make sure whether
> your data is twinned, it is better to take a look twinning test than seeing
> the R-values from refinement with twin operators. Applying twin operators
> always give you better R- values.
>
> Then, here are some questions related to my current analysis.
> Q1. As many of you concerned and I also had speculation on my C2 cell
> refinement with twin operators, I tried to analyze twinning of my datasets.
> (ATTACHED)
> As you can see the log file from truncate, I guess my crystal is twinned.
> In addition to this, I also followed the discussion between Randy and Lan.
> I wanted to make sure whether my refinement with twin operators is correct
> or not.  As Randy recommend and based on the paper from Garib N. Murshudov (
> http://www.ysbl.york.ac.uk/refmac/papers/Rfactor.pdf), I could see the
> drop of R-values with twin operators is always possible, however, in this
> paper, I could see the gap between refinements w/ or w/o twin operators is
> quite smaller than my case. (0.49-> 0.41 or 0.58 -> 0.52 vs my case 0.39 ->
> 0.23). Together, based on both twinning-test and good R-values, I now
> believe C2 refinement with twin operators are true. What do you think?
>
> Q2-1. Because some of you asked me about the original spots and
> re-indexing the data sets with high symmetry SG,  I went back to mosflm,
> and here are some images from my analysis. As you can clearly see there are
> strong/weak spots in the frames, and if you increase the threshold you can
> pick the strong spots (ATTACHED). Based on this, initially, I assumed that
> my crystal has tNCS, because I thought this strong - weak pattern is caused
> by tNCS. or Am I wrong?
>
> Q2-2. During new indexing, I choose two unit cells (large and small) and
> integrated them into 2 SG (P31 1 2 or P31 2 1) as Lijun commented. (four
> data sets)
> All of these data sets indicated twinning, while smaller one showed higher
> twinning fractions. Then, I ran phaser, and found that phaser only found
> the solution from P31 2 1SG from both large and small cells. However, I
> could see crash of molecules from large cell, and I decided to stick with
> small cell again. Then, during refinement with twin operators the R-values
> drops from (0.46/0.49) to (0.37/0.42), even the map looks already good. I
> assume that applying high symmetry SG is not possible for this case, and
> again C2 refinement might be correct.
>
> Q2-3. Related to Q1 and 2-1, Now, I think C2 (small cell) refinement with
> twin operator is right solution. Then, here I have another question.
> Based on the observations from frames, I guessed my crystal has tNCS, and
> I only followed strong spots. (Maybe I am wrong ?)
> Then, data sets with only strong spots suggests my crystal is twinned.
> Together, I am thinking my crystal has both tNCS and twinning, and I could
> solve the structure by following strong spots with twin operators.
>
> Again, I would like to express my special thanks to you all for giving me
> valuable comments.
> I hope this post will also useful for others in the future.
>
> Donghyuk
>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> 

Re: [ccp4bb] translational NCS & twinning

[Guan, Lan]

Dear Randy,
Thank you for the important information and the useful link.  I have another 
question to bother you.
How accurately can these twining assessment programs report the data?  What 
could prevent these programs from reporting twinning data? (Twinned data are 
reported as untwinned data)

 Thanks,

Lan




On Jan 12, 2019, at 6:19 AM, Randy Read 
mailto:rj...@cam.ac.uk>> wrote:

CAUTION: This email originated from outside of TTUHSC. Do not click links or 
open attachments unless you recognize the sender and know the content is safe.


Dear Lan,

Yes, that’s a serious problem that has led some people astray, including a few 
papers where people got apparently good R-factors by invoking non-existent 
twinning.

You can find a brief discussion of this point on the CCP4 wiki 
(https://urldefense.proofpoint.com/v2/url?u=https-3A__strucbio.biologie.uni-2Dkonstanz.de_ccp4wiki_index.php_R-2Dfactors=DwIFaQ=dIAUvHjI5lMnDD45iB3vgA=SZfWxbou0cinb5MH936uQKMweCFFe1qb2Aj8yA2JJ_E=Izy3-mP_RaC3MwlNJdICSKPXfwcW_MFeFmkKfem5tEo=ccGuy7JKlEPfv7NvBSM2aRt4EdZz6m-oEwgKBv9Bl4k=),
 which has links to a couple of publications where Garib Murshudov has worked 
out what happens to R-factors when you treat the data as twinned even when it 
isn’t.

Best wishes,

Randy

-
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 Jan 2019, at 23:05, Guan, Lan 
mailto:lan.g...@ttuhsc.edu>> wrote:

Hi Randy,


As Jacob and others have mentioned, you will always get lower R-factors once 
you treat the data as being twinned, and the more twin operators the bigger the 
reduction in R-factors.

Do normal data with no twinning, but refined with twin operator(s), show 
similar phenomenon?  If it decreases R-factors for normal data, how much it can 
achieve (5-10% lower)?

Thanks,


Lan



So you need very strong evidence, independent of R-factors, to invoke twinning. 
 In this case, the L-test should be reasonably trustworthy even in the presence 
of tNCS, and your L-test values are close to what one would expect for an 
untwinned crystal.  At most you have partial twinning, or perhaps twinning of a 
pseudo-symmetric crystal (a possibility Phil mentioned), where the effects on 
intensity statistics are reduced.

One way to address a problem like this is to solve the structure in a lower 
symmetry space group (as you have done), but then to check whether the MR 
solution actually obeys the higher symmetry.  You can do this by looking at the 
crystal packing or at merging statistics for Fcalcs after a refinement with 
highly restrained NCS.  A similar sort of analysis is automated in the Zanuda 
tool in CCP4.

Dealing with potential complications from combinations of twinning and 
pseudosymmetry is one of the more challenging aspects of crystallography, but 
it's a good learning experience.  Good luck!

Randy Read

On 10 Jan 2019, at 22:38, Donghyuk Shin 
mailto:sdh...@gmail.com>> wrote:

Dear all,

Thank you very much for all of your suggestions and sharing experiences.
As many of you commented, the current small unit cell C2 refinement seems to be 
incorrect or correct, and I should put some efforts to crack this question.

- To Phill Jeffrey,
The idea, trying to find high symmetry SG with small unit cell C2 data is good 
idea, and I will try this.
For your last comments, identifiable electron density differences between each 
chain,
I guess there should not be other densities between chains if my current SG and 
model is correct. Am I right?

- To Ethan,
Turning off the automatic_tNCS_option seems to be good option.
I think, my current data seems to be twinned then tNCS which I am not sure at 
this moment. But I will keep your advice in my mind.

- To Phoebe A. Rice,
It is quite interesting that you also could get structure solution by indexing 
strong spots and having smaller unit cell.
Actually, I was wondering how it was possible that having half-sized unit cell 
could have solution, while full-sized unit cell could not.
It will be great if you can share your experience a bit more (e.g the size of 
smaller unit cell used in initial search for both 1szp and 3pkz)

Again, thank you very much for all of your suggestion.

Best wishes,
Donghyuk



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Re: [ccp4bb] translational NCS & twinning

[Randy Read]

Dear Lan,

Yes, that’s a serious problem that has led some people astray, including a few 
papers where people got apparently good R-factors by invoking non-existent 
twinning.

You can find a brief discussion of this point on the CCP4 wiki 
(https://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/R-factors), which 
has links to a couple of publications where Garib Murshudov has worked out what 
happens to R-factors when you treat the data as twinned even when it isn’t.

Best wishes,

Randy

-
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 Jan 2019, at 23:05, Guan, Lan  wrote:
> 
> Hi Randy,
> 
> 
>> As Jacob and others have mentioned, you will always get lower R-factors once 
>> you treat the data as being twinned, and the more twin operators the bigger 
>> the reduction in R-factors.  
> 
> Do normal data with no twinning, but refined with twin operator(s), show 
> similar phenomenon?  If it decreases R-factors for normal data, how much it 
> can achieve (5-10% lower)?
> 
> Thanks,
> 
> 
> Lan
> 
> 
> 
>> So you need very strong evidence, independent of R-factors, to invoke 
>> twinning.  In this case, the L-test should be reasonably trustworthy even in 
>> the presence of tNCS, and your L-test values are close to what one would 
>> expect for an untwinned crystal.  At most you have partial twinning, or 
>> perhaps twinning of a pseudo-symmetric crystal (a possibility Phil 
>> mentioned), where the effects on intensity statistics are reduced.
>> 
>> One way to address a problem like this is to solve the structure in a lower 
>> symmetry space group (as you have done), but then to check whether the MR 
>> solution actually obeys the higher symmetry.  You can do this by looking at 
>> the crystal packing or at merging statistics for Fcalcs after a refinement 
>> with highly restrained NCS.  A similar sort of analysis is automated in the 
>> Zanuda tool in CCP4.
>> 
>> Dealing with potential complications from combinations of twinning and 
>> pseudosymmetry is one of the more challenging aspects of crystallography, 
>> but it's a good learning experience.  Good luck!
>> 
>> Randy Read
>> 
>>> On 10 Jan 2019, at 22:38, Donghyuk Shin  wrote:
>>> 
>>> Dear all,
>>> 
>>> Thank you very much for all of your suggestions and sharing experiences.
>>> As many of you commented, the current small unit cell C2 refinement seems 
>>> to be incorrect or correct, and I should put some efforts to crack this 
>>> question.
>>> 
>>> - To Phill Jeffrey,
>>> The idea, trying to find high symmetry SG with small unit cell C2 data is 
>>> good idea, and I will try this.
>>> For your last comments, identifiable electron density differences between 
>>> each chain,
>>> I guess there should not be other densities between chains if my current SG 
>>> and model is correct. Am I right?
>>> 
>>> - To Ethan,
>>> Turning off the automatic_tNCS_option seems to be good option.
>>> I think, my current data seems to be twinned then tNCS which I am not sure 
>>> at this moment. But I will keep your advice in my mind.
>>> 
>>> - To Phoebe A. Rice,
>>> It is quite interesting that you also could get structure solution by 
>>> indexing strong spots and having smaller unit cell.
>>> Actually, I was wondering how it was possible that having half-sized unit 
>>> cell could have solution, while full-sized unit cell could not.
>>> It will be great if you can share your experience a bit more (e.g the size 
>>> of smaller unit cell used in initial search for both 1szp and 3pkz)
>>> 
>>> Again, thank you very much for all of your suggestion.
>>> 
>>> Best wishes,
>>> Donghyuk
>>> 
>>> 
>>> 
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwIFAg=dIAUvHjI5lMnDD45iB3vgA=SZfWxbou0cinb5MH936uQKMweCFFe1qb2Aj8yA2JJ_E=1tRkoB-BHhBWDCtbPikRHJfYIDIqpaGbVoF5xFjTLD4=007v9D7bqs0h2uVFwau-m4cmT4PVlWyYZ2UVxJvWkrs=
>> 
>> --
>> 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
>> 
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> 

Re: [ccp4bb] translational NCS & twinning

[Guan, Lan]

Hi Randy,


> As Jacob and others have mentioned, you will always get lower R-factors once 
> you treat the data as being twinned, and the more twin operators the bigger 
> the reduction in R-factors.  

Do normal data with no twinning, but refined with twin operator(s), show 
similar phenomenon?  If it decreases R-factors for normal data, how much it can 
achieve (5-10% lower)?

Thanks,


Lan



> So you need very strong evidence, independent of R-factors, to invoke 
> twinning.  In this case, the L-test should be reasonably trustworthy even in 
> the presence of tNCS, and your L-test values are close to what one would 
> expect for an untwinned crystal.  At most you have partial twinning, or 
> perhaps twinning of a pseudo-symmetric crystal (a possibility Phil 
> mentioned), where the effects on intensity statistics are reduced.
> 
> One way to address a problem like this is to solve the structure in a lower 
> symmetry space group (as you have done), but then to check whether the MR 
> solution actually obeys the higher symmetry.  You can do this by looking at 
> the crystal packing or at merging statistics for Fcalcs after a refinement 
> with highly restrained NCS.  A similar sort of analysis is automated in the 
> Zanuda tool in CCP4.
> 
> Dealing with potential complications from combinations of twinning and 
> pseudosymmetry is one of the more challenging aspects of crystallography, but 
> it's a good learning experience.  Good luck!
> 
> Randy Read
> 
>> On 10 Jan 2019, at 22:38, Donghyuk Shin  wrote:
>> 
>> Dear all,
>> 
>> Thank you very much for all of your suggestions and sharing experiences.
>> As many of you commented, the current small unit cell C2 refinement seems to 
>> be incorrect or correct, and I should put some efforts to crack this 
>> question.
>> 
>> - To Phill Jeffrey,
>> The idea, trying to find high symmetry SG with small unit cell C2 data is 
>> good idea, and I will try this.
>> For your last comments, identifiable electron density differences between 
>> each chain,
>> I guess there should not be other densities between chains if my current SG 
>> and model is correct. Am I right?
>> 
>> - To Ethan,
>> Turning off the automatic_tNCS_option seems to be good option.
>> I think, my current data seems to be twinned then tNCS which I am not sure 
>> at this moment. But I will keep your advice in my mind.
>> 
>> - To Phoebe A. Rice,
>> It is quite interesting that you also could get structure solution by 
>> indexing strong spots and having smaller unit cell.
>> Actually, I was wondering how it was possible that having half-sized unit 
>> cell could have solution, while full-sized unit cell could not.
>> It will be great if you can share your experience a bit more (e.g the size 
>> of smaller unit cell used in initial search for both 1szp and 3pkz)
>> 
>> Again, thank you very much for all of your suggestion.
>> 
>> Best wishes,
>> Donghyuk
>> 
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwIFAg=dIAUvHjI5lMnDD45iB3vgA=SZfWxbou0cinb5MH936uQKMweCFFe1qb2Aj8yA2JJ_E=1tRkoB-BHhBWDCtbPikRHJfYIDIqpaGbVoF5xFjTLD4=007v9D7bqs0h2uVFwau-m4cmT4PVlWyYZ2UVxJvWkrs=
> 
> --
> 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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Re: [ccp4bb] translational NCS & twinning

[lijunliuks]

Donghyuk:

How good were your original images?  If the paeks are sharp and clean, your processing could be easier; or you need more efforts.  The data processing statistics is more important for troubleshooting.

Your showed an apparent SG of p6322, but it does have to be exact, especially when with various NCS.  XTRIAGE output the list of 6(1) related systematic absences but none of them went above 10 I/sig(I),  althought not atypical, have you checked the I's against their neighboring I(0 0 6n) to confirm?  Did you try all possible P6i22 within your phaser search?

With twinning test, the closest low symmetry test would be for P6i and P3i21   and P3i12, the later 2 you seemed to have skipped?  High odered twinning in P3i could be possible but would be hard to deal with.

A C2 lattice could be reindexed to a new one with halved a and halved b, which is possible, but if the new one were still with C2, then it would be cautious.

Lijun Liu
DLX Scientific




发自我的小米手机在 Donghyuk Shin ，2019年1月10日 上午4:22写道：Dear all,

I am having tough time with my Xtal data sets those seem to be twinned or have translational NCS, and it will be greatly appreciated if you can give me some advices or comments!

Data was initially processed with XDS and scaled with aimless without specifying certain space group (SG).
Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates there is non-origin peak after patterson analysis. (attached log)
And, I could not get the proper MR-solution from this data sets.

Because I read that twinning and tNCS cannot be properly distinguished at high SG, I went down to subgroup either P32 or P6 assuming that there is twinning which make data set seems to have apparently high SG. (procedure was same XDS->aimless but I specified the SG to keep them)
Then, xtriage still indicates there is non-origin peak as before, but found twin laws for the data sets (attached log).
However, I still could not get the right MR-solution.
Then, I went even further down to P3 or C2, and xtriage found more twin laws which is make sense because of the lower SG. (attached log)
Again, I could not get the MR-solution.
For all the MR running above, I assumed that phaser(ccp4 module) automatically applied tNCS if they present. or should I have to tick on button in the expert parameters?

Then, I went back to the image and processed the datasets with mosflm by checking the indexed spots.
During this step, I played with the threshold for indexing to follow the strong spot for get correct SG.
I am not sure whether this is correct or not, but by putting high threshold for indexing (e.g. ~15) I could index the data with C2 which has half dimension for a,b axes (116.348,  67.218, 182.861,  90, 90, 90) to the original unit cell (232.533, 134.202, 182.67, 90, 90, 90).
With this, I could put 3 molecules in ASU by MR. During refinement, I felt that the R values were not dropping, and I applied twin refinement.
without twin refinement the R values were (0.39/0.44, work/free), and applying twin refinement gave me significantly better values (0.23/0.26).
Because there were 6 twin operators which may cause this huge R value drop, I speculate whether this is true or not.

Your comments will be greatly helpful! 

With you all the best,
Donghyuk





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[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning

[IGBMC]

Le Vendredi 11 Janvier 2019 12:18 CET,  a écrit:

Thank you Herman you for this clear and informative comments !!
Particularly your last point, which I found very instructive.
Now I remember the title of the article by T.O. Yeates "Protein crystals and 
their evil twins" in 1999. And this corresponds well to the difficulty of the 
problem.
Philippe D.

Philippe Dumas

> Dear Philippe,
>
> As Randy just pointed out, when twinning, pseudosymmetry and other 
> pathologies come into play, things really get complicated.
> I agree with what you said but for the current problem, things may be more 
> complicated.
>
> To summarize:
> - "bona fide" twinning: there are two different, intergrown crystals and the 
> intensities of both crystals just add up.
> - "nano scale" twinning, called statistical disorder: the two orientations 
> are randomly distributed through the crystals and the diffraction of both 
> conformations interferes. The conformations behave like alternate 
> conformations.
> - Twinning/pseudosymmetry/wrong unit cell etc.: Here the two conformations, 
> present in the large (true) unit cell and related by crystallographic or 
> noncrystallographic symmetry, may not fit in the small (false) unit cell and 
> be accounted for by introducing twinning where none is present. Especially 
> with 6 twinning operators, the refinement programs have a lot of room to 
> tweak around to reduce the R-factors.
>
> Therefore my advice would be:
> - Critically check the space group and especially how weak are the weak 
> reflections discarded with the small unit cell?
> - the solution you got in the small unit cell may be a subset of what is 
> present in the large unit cell, so I would also try molecular replacement 
> with the ensemble of molecules you got in the small unit cell.
>
> My two cents,
> Herman
>
>
> -Ursprüngliche Nachricht-
> Von: DUMAS Philippe (IGBMC) [mailto:p.du...@ibmc-cnrs.unistra.fr]
> Gesendet: Freitag, 11. Januar 2019 11:52
> An: Schreuder, Herman /DE
> Cc: CCP4BB@JISCMAIL.AC.UK
> Betreff: Re: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS 
> & twinning
>
>
> Le Vendredi 11 Janvier 2019 09:07 CET, herman.schreu...@sanofi.com a écrit:
>
> Dear Herman,
> As far as I understood the twinning problem, what you say is only true in 
> some occasions, and not in others.
> If the "macroscopic" domains are so small that they are smaller than the 
> X-rays coherence length, then you may do what you say because the X-rays 
> emitted by the different domains can interfere.
> But, if the domains become large, the X-rays emitted by the different domains 
> do not interfere anymore and you have to add the weighted intensities, not 
> the amplitudes, of each domain.
> I hope I understood well your comment and did not "interfere negatively" with 
> the thread...
> Best
> Philippe Dumas
>
> > Dear Lan,
> >
> > Thank you for your compliment. I do not use Xtriage, so I did not bother 
> > looking at the log files.
> >
> > What I meant to say is that with twinning, the crystal has different 
> > macroscopic domains where the molecules have different orientations, say 
> > one domain with orientation A and one domain with orientation B. Since 
> > these domains grow on top of each other, they are usually related by a twin 
> > operator similar to a crystallographic operator such as a twofold axis.
> > The fourier transform of the electron density of the crystal is the 
> > convolution of the fourier transform of the individual molecules with the 
> > crystal lattice, with the fourier transform of the individual molecules 
> > usually giving the stronger contribution. So to get a solution with a 
> > decent R-factor, one must include all orientations (A, B etc.) in the 
> > model, with the position of the molecules in the crystal lattice 
> > contributing less to the diffraction pattern. So one can put the 
> > orientations on top of each other in a small unit cell using twinning, or 
> > put them in a larger unit cell at different positions using 
> > crystallographic or non-crystallographic symmetry. That is what I meant be 
> > "twinning" (N)CS.
> >
> > Hope this makes my remark a little clearer.
> >
> > Best,
> > Herman
> >
> > PS: While other BB readers may have had the same question, I have posted 
> > the reply to the BB. I hope you don't mind.
> >
> >
> > -Ursprüngliche Nachricht-
> > Von: Guan, Lan [mailto:lan.g...@ttuhsc.edu]
> > Gesendet: Donnerstag, 10. Januar 2019 20:53
> > An: Schreuder, Herman /DE
> > Betreff: 

[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning

[Herman . Schreuder]

Dear Philippe,
 
As Randy just pointed out, when twinning, pseudosymmetry and other pathologies 
come into play, things really get complicated.
I agree with what you said but for the current problem, things may be more 
complicated. 

To summarize:
- "bona fide" twinning: there are two different, intergrown crystals and the 
intensities of both crystals just add up.
- "nano scale" twinning, called statistical disorder: the two orientations are 
randomly distributed through the crystals and the diffraction of both 
conformations interferes. The conformations behave like alternate conformations.
- Twinning/pseudosymmetry/wrong unit cell etc.: Here the two conformations, 
present in the large (true) unit cell and related by crystallographic or 
noncrystallographic symmetry, may not fit in the small (false) unit cell and be 
accounted for by introducing twinning where none is present. Especially with 6 
twinning operators, the refinement programs have a lot of room to tweak around 
to reduce the R-factors.

Therefore my advice would be:
- Critically check the space group and especially how weak are the weak 
reflections discarded with the small unit cell?
- the solution you got in the small unit cell may be a subset of what is 
present in the large unit cell, so I would also try molecular replacement with 
the ensemble of molecules you got in the small unit cell.

My two cents,
Herman


-Ursprüngliche Nachricht-
Von: DUMAS Philippe (IGBMC) [mailto:p.du...@ibmc-cnrs.unistra.fr] 
Gesendet: Freitag, 11. Januar 2019 11:52
An: Schreuder, Herman /DE
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & 
twinning


Le Vendredi 11 Janvier 2019 09:07 CET, herman.schreu...@sanofi.com a écrit:

Dear Herman,
As far as I understood the twinning problem, what you say is only true in some 
occasions, and not in others.
If the "macroscopic" domains are so small that they are smaller than the X-rays 
coherence length, then you may do what you say because the X-rays emitted by 
the different domains can interfere.
But, if the domains become large, the X-rays emitted by the different domains 
do not interfere anymore and you have to add the weighted intensities, not the 
amplitudes, of each domain.
I hope I understood well your comment and did not "interfere negatively" with 
the thread...
Best
Philippe Dumas

> Dear Lan,
>
> Thank you for your compliment. I do not use Xtriage, so I did not bother 
> looking at the log files.
>
> What I meant to say is that with twinning, the crystal has different 
> macroscopic domains where the molecules have different orientations, say one 
> domain with orientation A and one domain with orientation B. Since these 
> domains grow on top of each other, they are usually related by a twin 
> operator similar to a crystallographic operator such as a twofold axis.
> The fourier transform of the electron density of the crystal is the 
> convolution of the fourier transform of the individual molecules with the 
> crystal lattice, with the fourier transform of the individual molecules 
> usually giving the stronger contribution. So to get a solution with a decent 
> R-factor, one must include all orientations (A, B etc.) in the model, with 
> the position of the molecules in the crystal lattice contributing less to the 
> diffraction pattern. So one can put the orientations on top of each other in 
> a small unit cell using twinning, or put them in a larger unit cell at 
> different positions using crystallographic or non-crystallographic symmetry. 
> That is what I meant be "twinning" (N)CS.
>
> Hope this makes my remark a little clearer.
>
> Best,
> Herman
>
> PS: While other BB readers may have had the same question, I have posted the 
> reply to the BB. I hope you don't mind.
>
>
> -Ursprüngliche Nachricht-
> Von: Guan, Lan [mailto:lan.g...@ttuhsc.edu]
> Gesendet: Donnerstag, 10. Januar 2019 20:53
> An: Schreuder, Herman /DE
> Betreff: Re: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning
>
> Dear Herman,
> I have read your insightful comments on twining and tNCS for years now, which 
> is very useful and helpful!  Thanks,
>
> For Donghyuk’s case, do you think that he really has a twinning issue?  With 
> a lower symmetry, all possible twining operators is always reported in the 
> Xtriage and did not mean a real twin existed.  His L test shows a twin 
> fraction of 0.00 in his log file.  The intensity statistics does not really 
> indicate an actually twinning.  Base on the refinement, twin law is needed to 
> get refinement going.  It looks like a twin.  I am confused...
>
> > the molecules are related by "twinning" (N)CS?
>
>
> What does this mean “ twinning (N)CS"?  Would you p

Re: [ccp4bb] translational NCS & twinning

[Eleanor Dodson]

That P6322 Xtriage shows pretty clearly that you have good data, and that
it is not twinned..

So I think you stick with that  spacegroup - introducing false twin laws to
explain true crystal symmetry reduces the R values at the expense of
getting clear electron density.

You could process the data in a cell with the a b axes halved, choosing
only the strong reflections not affected by the non-cryst Patterson vector,
but in fact the MR programs take such a translation into account during
structure solution .

Eleanor Dodson




On Fri, 11 Jan 2019 at 10:54, Randy Read  wrote:

> Hi,
>
> I'm just catching up on the BB after the CCP4 Study Weekend!
>
> Going back to the beginning of this thread, it's true that tNCS and
> twinning have opposite effects on the intensity moments, which can mask
> each other.  However, for simple cases (as this one appears to be) with a
> single NCS translation, the tNCS analysis in Phaser seems to be pretty
> successful at deconvoluting those effects.  It would be interesting to see
> the overall second moments and the second moments as a function of
> resolution from a run in Phaser, either in NCS mode or in MR_AUTO mode.
>
> In such a case, once the tNCS is properly characterised, Phaser places
> molecules in pairs (or higher multiples in more complex cases of
> higher-order tNCS).  That should deal automatically with cases like the
> ones Phoebe mentions, where you could temporarily reduce the cell by a
> factor of two, but it works more generally when the tNCS doesn't correspond
> to a cell doubling.  We'd appreciate hearing about any cases where
> reindexing in a smaller cell works and Phaser's automated treatment
> didn't!  Or, indeed, about cases like the one Ethan mentioned where turning
> off the automatic tNCS correction helped.  (I've already been in touch with
> Ethan off-line.)
>
> As Jacob and others have mentioned, you will always get lower R-factors
> once you treat the data as being twinned, and the more twin operators the
> bigger the reduction in R-factors.  So you need very strong evidence,
> independent of R-factors, to invoke twinning.  In this case, the L-test
> should be reasonably trustworthy even in the presence of tNCS, and your
> L-test values are close to what one would expect for an untwinned crystal.
> At most you have partial twinning, or perhaps twinning of a
> pseudo-symmetric crystal (a possibility Phil mentioned), where the effects
> on intensity statistics are reduced.
>
> One way to address a problem like this is to solve the structure in a
> lower symmetry space group (as you have done), but then to check whether
> the MR solution actually obeys the higher symmetry.  You can do this by
> looking at the crystal packing or at merging statistics for Fcalcs after a
> refinement with highly restrained NCS.  A similar sort of analysis is
> automated in the Zanuda tool in CCP4.
>
> Dealing with potential complications from combinations of twinning and
> pseudosymmetry is one of the more challenging aspects of crystallography,
> but it's a good learning experience.  Good luck!
>
> Randy Read
>
> > On 10 Jan 2019, at 22:38, Donghyuk Shin  wrote:
> >
> > Dear all,
> >
> > Thank you very much for all of your suggestions and sharing experiences.
> > As many of you commented, the current small unit cell C2 refinement
> seems to be incorrect or correct, and I should put some efforts to crack
> this question.
> >
> > - To Phill Jeffrey,
> > The idea, trying to find high symmetry SG with small unit cell C2 data
> is good idea, and I will try this.
> > For your last comments, identifiable electron density differences
> between each chain,
> > I guess there should not be other densities between chains if my current
> SG and model is correct. Am I right?
> >
> > - To Ethan,
> > Turning off the automatic_tNCS_option seems to be good option.
> > I think, my current data seems to be twinned then tNCS which I am not
> sure at this moment. But I will keep your advice in my mind.
> >
> > - To Phoebe A. Rice,
> > It is quite interesting that you also could get structure solution by
> indexing strong spots and having smaller unit cell.
> > Actually, I was wondering how it was possible that having half-sized
> unit cell could have solution, while full-sized unit cell could not.
> > It will be great if you can share your experience a bit more (e.g the
> size of smaller unit cell used in initial search for both 1szp and 3pkz)
> >
> > Again, thank you very much for all of your suggestion.
> >
> > Best wishes,
> > Donghyuk
> >
> > 
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
> --
> 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 Road

Re: [ccp4bb] translational NCS & twinning

[Randy Read]

Hi,

I'm just catching up on the BB after the CCP4 Study Weekend!

Going back to the beginning of this thread, it's true that tNCS and twinning 
have opposite effects on the intensity moments, which can mask each other.  
However, for simple cases (as this one appears to be) with a single NCS 
translation, the tNCS analysis in Phaser seems to be pretty successful at 
deconvoluting those effects.  It would be interesting to see the overall second 
moments and the second moments as a function of resolution from a run in 
Phaser, either in NCS mode or in MR_AUTO mode.

In such a case, once the tNCS is properly characterised, Phaser places 
molecules in pairs (or higher multiples in more complex cases of higher-order 
tNCS).  That should deal automatically with cases like the ones Phoebe 
mentions, where you could temporarily reduce the cell by a factor of two, but 
it works more generally when the tNCS doesn't correspond to a cell doubling.  
We'd appreciate hearing about any cases where reindexing in a smaller cell 
works and Phaser's automated treatment didn't!  Or, indeed, about cases like 
the one Ethan mentioned where turning off the automatic tNCS correction helped. 
 (I've already been in touch with Ethan off-line.)

As Jacob and others have mentioned, you will always get lower R-factors once 
you treat the data as being twinned, and the more twin operators the bigger the 
reduction in R-factors.  So you need very strong evidence, independent of 
R-factors, to invoke twinning.  In this case, the L-test should be reasonably 
trustworthy even in the presence of tNCS, and your L-test values are close to 
what one would expect for an untwinned crystal.  At most you have partial 
twinning, or perhaps twinning of a pseudo-symmetric crystal (a possibility Phil 
mentioned), where the effects on intensity statistics are reduced.

One way to address a problem like this is to solve the structure in a lower 
symmetry space group (as you have done), but then to check whether the MR 
solution actually obeys the higher symmetry.  You can do this by looking at the 
crystal packing or at merging statistics for Fcalcs after a refinement with 
highly restrained NCS.  A similar sort of analysis is automated in the Zanuda 
tool in CCP4.

Dealing with potential complications from combinations of twinning and 
pseudosymmetry is one of the more challenging aspects of crystallography, but 
it's a good learning experience.  Good luck!

Randy Read

> On 10 Jan 2019, at 22:38, Donghyuk Shin  wrote:
> 
> Dear all,
> 
> Thank you very much for all of your suggestions and sharing experiences.
> As many of you commented, the current small unit cell C2 refinement seems to 
> be incorrect or correct, and I should put some efforts to crack this question.
> 
> - To Phill Jeffrey, 
> The idea, trying to find high symmetry SG with small unit cell C2 data is 
> good idea, and I will try this.
> For your last comments, identifiable electron density differences between 
> each chain, 
> I guess there should not be other densities between chains if my current SG 
> and model is correct. Am I right?
> 
> - To Ethan,
> Turning off the automatic_tNCS_option seems to be good option.
> I think, my current data seems to be twinned then tNCS which I am not sure at 
> this moment. But I will keep your advice in my mind.
> 
> - To Phoebe A. Rice,
> It is quite interesting that you also could get structure solution by 
> indexing strong spots and having smaller unit cell.
> Actually, I was wondering how it was possible that having half-sized unit 
> cell could have solution, while full-sized unit cell could not.
> It will be great if you can share your experience a bit more (e.g the size of 
> smaller unit cell used in initial search for both 1szp and 3pkz)
> 
> Again, thank you very much for all of your suggestion.
> 
> Best wishes,
> Donghyuk
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

--
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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Re: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning

[IGBMC]

Le Vendredi 11 Janvier 2019 09:07 CET, herman.schreu...@sanofi.com a écrit:

Dear Herman,
As far as I understood the twinning problem, what you say is only true in some 
occasions, and not in others.
If the "macroscopic" domains are so small that they are smaller than the X-rays 
coherence length, then you may do what you say because the X-rays emitted by 
the different domains can interfere.
But, if the domains become large, the X-rays emitted by the different domains 
do not interfere anymore and you have to add the weighted intensities, not the 
amplitudes, of each domain.
I hope I understood well your comment and did not "interfere negatively" with 
the thread...
Best
Philippe Dumas

> Dear Lan,
>
> Thank you for your compliment. I do not use Xtriage, so I did not bother 
> looking at the log files.
>
> What I meant to say is that with twinning, the crystal has different 
> macroscopic domains where the molecules have different orientations, say one 
> domain with orientation A and one domain with orientation B. Since these 
> domains grow on top of each other, they are usually related by a twin 
> operator similar to a crystallographic operator such as a twofold axis.
> The fourier transform of the electron density of the crystal is the 
> convolution of the fourier transform of the individual molecules with the 
> crystal lattice, with the fourier transform of the individual molecules 
> usually giving the stronger contribution. So to get a solution with a decent 
> R-factor, one must include all orientations (A, B etc.) in the model, with 
> the position of the molecules in the crystal lattice contributing less to the 
> diffraction pattern. So one can put the orientations on top of each other in 
> a small unit cell using twinning, or put them in a larger unit cell at 
> different positions using crystallographic or non-crystallographic symmetry. 
> That is what I meant be "twinning" (N)CS.
>
> Hope this makes my remark a little clearer.
>
> Best,
> Herman
>
> PS: While other BB readers may have had the same question, I have posted the 
> reply to the BB. I hope you don't mind.
>
>
> -Ursprüngliche Nachricht-
> Von: Guan, Lan [mailto:lan.g...@ttuhsc.edu]
> Gesendet: Donnerstag, 10. Januar 2019 20:53
> An: Schreuder, Herman /DE
> Betreff: Re: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning
>
> Dear Herman,
> I have read your insightful comments on twining and tNCS for years now, which 
> is very useful and helpful!  Thanks,
>
> For Donghyuk’s case, do you think that he really has a twinning issue?  With 
> a lower symmetry, all possible twining operators is always reported in the 
> Xtriage and did not mean a real twin existed.  His L test shows a twin 
> fraction of 0.00 in his log file.  The intensity statistics does not really 
> indicate an actually twinning.  Base on the refinement, twin law is needed to 
> get refinement going.  It looks like a twin.  I am confused...
>
> > the molecules are related by "twinning" (N)CS?
>
>
> What does this mean “ twinning (N)CS"?  Would you please kindly explain it 
> further?
>
> Thanks,
>
>
> Lan
>
>
> 
> Lan Guan, MD PhD
> Associate Professor | Department of Cell Physiology and Molecular Biophysics
> Director | Center for Membrane Protein Research
>
> 3601 4th St. MS 6551 | Lubbock, TX 79430
> 5A148A (Office) | (1) 806 743-3102 (Phone) | lan.g...@ttuhsc.edu (E-Mail)
>
> http://www.ttuhsc.edu/medicine/cell-physiology-molecular-biophysics/faculty/guan/
> https://www.ttuhsc.edu/centers-institutes/membrane-protein-research/

> 
>
> > On Jan 10, 2019, at 11:10 AM, herman.schreu...@sanofi.com wrote:
> >
> > CAUTION: This email originated from outside of TTUHSC. Do not click links 
> > or open attachments unless you recognize the sender and know the content is 
> > safe.
> >
> >
> > Dear Donghyuk,
> >
> > Unfortunately, everything is possible when NCS, twinning etc. get into the 
> > game. I do not have answers, but some questions for you to think about:
> > - Do you really have 6 twinning operators, or only one operator and are the 
> > other operators generated by (non)crystallographic symmetry?
> > - Both your P6322 cell and your C2 cell have angles of 90 90 90. For P6322, 
> > the last angle should be 120 and for C2, only the second angle is 
> > constrained to be 90. Maybe you should check that not somewhere something 
> > went wrong with the cell angles.
> > - How weak are the reflecti

[ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning

[Herman . Schreuder]

Dear Lan,

Thank you for your compliment. I do not use Xtriage, so I did not bother 
looking at the log files.

What I meant to say is that with twinning, the crystal has different 
macroscopic domains where the molecules have different orientations, say one 
domain with orientation A and one domain with orientation B. Since these 
domains grow on top of each other, they are usually related by a twin operator 
similar to a crystallographic operator such as a twofold axis. 
The fourier transform of the electron density of the crystal is the convolution 
of the fourier transform of the individual molecules with the crystal lattice, 
with the fourier transform of the individual molecules usually giving the 
stronger contribution. So to get a solution with a decent R-factor, one must 
include all orientations (A, B etc.) in the model, with the position of the 
molecules in the crystal lattice contributing less to the diffraction pattern. 
So one can put the orientations on top of each other in a small unit cell using 
twinning, or put them in a larger unit cell at different positions using 
crystallographic or non-crystallographic symmetry. That is what I meant be 
"twinning" (N)CS.

Hope this makes my remark a little clearer.

Best,
Herman

PS: While other BB readers may have had the same question, I have posted the 
reply to the BB. I hope you don't mind.


-Ursprüngliche Nachricht-
Von: Guan, Lan [mailto:lan.g...@ttuhsc.edu] 
Gesendet: Donnerstag, 10. Januar 2019 20:53
An: Schreuder, Herman /DE
Betreff: Re: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning

Dear Herman,
I have read your insightful comments on twining and tNCS for years now, which 
is very useful and helpful!  Thanks,

For Donghyuk’s case, do you think that he really has a twinning issue?  With a 
lower symmetry, all possible twining operators is always reported in the 
Xtriage and did not mean a real twin existed.  His L test shows a twin fraction 
of 0.00 in his log file.  The intensity statistics does not really indicate an 
actually twinning.  Base on the refinement, twin law is needed to get 
refinement going.  It looks like a twin.  I am confused...

> the molecules are related by "twinning" (N)CS?


What does this mean “ twinning (N)CS"?  Would you please kindly explain it 
further?

Thanks,


Lan



Lan Guan, MD PhD
Associate Professor | Department of Cell Physiology and Molecular Biophysics
Director | Center for Membrane Protein Research

3601 4th St. MS 6551 | Lubbock, TX 79430
5A148A (Office) | (1) 806 743-3102 (Phone) | lan.g...@ttuhsc.edu (E-Mail)

http://www.ttuhsc.edu/medicine/cell-physiology-molecular-biophysics/faculty/guan/
https://www.ttuhsc.edu/centers-institutes/membrane-protein-research/


> On Jan 10, 2019, at 11:10 AM, herman.schreu...@sanofi.com wrote:
> 
> CAUTION: This email originated from outside of TTUHSC. Do not click links or 
> open attachments unless you recognize the sender and know the content is safe.
> 
> 
> Dear Donghyuk,
> 
> Unfortunately, everything is possible when NCS, twinning etc. get into the 
> game. I do not have answers, but some questions for you to think about:
> - Do you really have 6 twinning operators, or only one operator and are the 
> other operators generated by (non)crystallographic symmetry?
> - Both your P6322 cell and your C2 cell have angles of 90 90 90. For P6322, 
> the last angle should be 120 and for C2, only the second angle is constrained 
> to be 90. Maybe you should check that not somewhere something went wrong with 
> the cell angles.
> - How weak are the reflections that got discarded by halving the a- and 
> b-axes? Do they have significant intensity, or is it only noise?
> - By shrinking the unit cell, you may have created artificial twinning when 
> in the large unit cell the molecules are related by "twinning" (N)CS.
> - Since you seem to have found a solution with the small unit cell, you could 
> see if you could fit this solution in the large unit cell: Process in P1 in 
> the large unit cell and use the ensemble (the complete! unit cell of your C2 
> solution, as a search model.
> - Your current solution maybe correct after all, but I would analyze it very 
> critically.
> 
> Best,
> Herman
> 
> 
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
> Donghyuk Shin
> Gesendet: Donnerstag, 10. Januar 2019 11:12
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: [EXTERNAL] [ccp4bb] translational NCS & twinning
> 
> Dear all,
> 
> I am having tough time with my Xtal data sets those seem to be twinned or 
> have translational NCS, and it will be grea

Re: [ccp4bb] translational NCS & twinning

[Donghyuk Shin]

Dear all,

Thank you very much for all of your suggestions and sharing experiences.
As many of you commented, the current small unit cell C2 refinement seems to be 
incorrect or correct, and I should put some efforts to crack this question.

- To Phill Jeffrey, 
The idea, trying to find high symmetry SG with small unit cell C2 data is good 
idea, and I will try this.
For your last comments, identifiable electron density differences between each 
chain, 
I guess there should not be other densities between chains if my current SG and 
model is correct. Am I right?

- To Ethan,
Turning off the automatic_tNCS_option seems to be good option.
I think, my current data seems to be twinned then tNCS which I am not sure at 
this moment. But I will keep your advice in my mind.

- To Phoebe A. Rice,
It is quite interesting that you also could get structure solution by indexing 
strong spots and having smaller unit cell.
Actually, I was wondering how it was possible that having half-sized unit cell 
could have solution, while full-sized unit cell could not.
It will be great if you can share your experience a bit more (e.g the size of 
smaller unit cell used in initial search for both 1szp and 3pkz)

Again, thank you very much for all of your suggestion.

Best wishes,
Donghyuk



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Re: [ccp4bb] translational NCS & twinning

[Phoebe A. Rice]

For two projects in the distant past, we dealt with tNCS by initially telling 
lies to the software (1szp and 3pkz).  The tNCS was strong enough that there 
was a clear weak/dark pattern in the diffraction pattern, so for the initial 
molecular replacement we used a data set in the smaller unit cell where only 
the strong spots had been boxed and reduced.  Then we moved that model to the 
proper, full unit cell (weak spots included) data set and finished the 
molecular replacement either by manually pushing the model around according to 
the appropriate vector(s) or just doing more molrep.

If you're desperate, and your weak spots are weak enough, it is worth a try!

~~~
Phoebe A. Rice
Dept. of Biochem & Mol. Biol. and
  Committee on Microbiology
https://voices.uchicago.edu/phoebericelab/
 
 

On 1/10/19, 12:18 PM, "CCP4 bulletin board on behalf of Ethan A Merritt" 
 wrote:

On Thursday, January 10, 2019 2:11:52 AM PST Donghyuk Shin wrote:
> Dear all,
> 
> I am having tough time with my Xtal data sets those seem to be twinned or 
have translational NCS, and it will be greatly appreciated if you can give me 
some advices or comments!
> 
> Data was initially processed with XDS and scaled with aimless without 
specifying certain space group (SG).
> Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates 
there is non-origin peak after patterson analysis. (attached log)
> And, I could not get the proper MR-solution from this data sets.
> 
> Because I read that twinning and tNCS cannot be properly distinguished at 
high SG, I went down to subgroup either P32 or P6 assuming that there is 
twinning which make data set seems to have apparently high SG. (procedure was 
same XDS->aimless but I specified the SG to keep them)
> Then, xtriage still indicates there is non-origin peak as before, but 
found twin laws for the data sets (attached log).
> However, I still could not get the right MR-solution.
> Then, I went even further down to P3 or C2, and xtriage found more twin 
laws which is make sense because of the lower SG. (attached log)

> Again, I could not get the MR-solution.
> For all the MR running above, I assumed that phaser(ccp4 module) 
automatically applied tNCS if they present. or should I have to tick on button 
in the expert parameters?
   ^^^

Hi Donghuk,

This may indeed be a possible source of problems.   I have recent 
experience with a
case where aimless/xtriage/phaser all report a large tNCS component, but 
allowing
phaser to use this information prevents finding a correct MR solution.  I 
can only
obtain the known correct solution by telling phaser explicitly _not_ to use 
tNCS.
In my case the true space group is P1 but the angles are all close to 90., 
causing
most indexing programs to falsely identify it as P2.  I cannot say how or 
why the
tNCS test triggers, but the known correct solution in P1 does not contain 
tNCS.

Ethan


> 
> Then, I went back to the image and processed the datasets with mosflm by 
checking the indexed spots.
> During this step, I played with the threshold for indexing to follow the 
strong spot for get correct SG.
> I am not sure whether this is correct or not, but by putting high 
threshold for indexing (e.g. ~15) I could index the data with C2 which has half 
dimension for a,b axes (116.348,  67.218, 182.861,  90, 90, 90) to the original 
unit cell (232.533, 134.202, 182.67, 90, 90, 90).
> With this, I could put 3 molecules in ASU by MR. During refinement, I 
felt that the R values were not dropping, and I applied twin refinement.
> without twin refinement the R values were (0.39/0.44, work/free), and 
applying twin refinement gave me significantly better values (0.23/0.26).
> Because there were 6 twin operators which may cause this huge R value 
drop, I speculate whether this is true or not.
> 
> Your comments will be greatly helpful! 
> 
> With you all the best,
> Donghyuk
> 
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1


-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742



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Re: [ccp4bb] translational NCS & twinning

[Ethan A Merritt]

On Thursday, January 10, 2019 2:11:52 AM PST Donghyuk Shin wrote:
> Dear all,
> 
> I am having tough time with my Xtal data sets those seem to be twinned or 
> have translational NCS, and it will be greatly appreciated if you can give me 
> some advices or comments!
> 
> Data was initially processed with XDS and scaled with aimless without 
> specifying certain space group (SG).
> Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates there 
> is non-origin peak after patterson analysis. (attached log)
> And, I could not get the proper MR-solution from this data sets.
> 
> Because I read that twinning and tNCS cannot be properly distinguished at 
> high SG, I went down to subgroup either P32 or P6 assuming that there is 
> twinning which make data set seems to have apparently high SG. (procedure was 
> same XDS->aimless but I specified the SG to keep them)
> Then, xtriage still indicates there is non-origin peak as before, but found 
> twin laws for the data sets (attached log).
> However, I still could not get the right MR-solution.
> Then, I went even further down to P3 or C2, and xtriage found more twin laws 
> which is make sense because of the lower SG. (attached log)

> Again, I could not get the MR-solution.
> For all the MR running above, I assumed that phaser(ccp4 module) 
> automatically applied tNCS if they present. or should I have to tick on 
> button in the expert parameters?
   ^^^

Hi Donghuk,

This may indeed be a possible source of problems.   I have recent experience 
with a
case where aimless/xtriage/phaser all report a large tNCS component, but 
allowing
phaser to use this information prevents finding a correct MR solution.  I can 
only
obtain the known correct solution by telling phaser explicitly _not_ to use 
tNCS.
In my case the true space group is P1 but the angles are all close to 90., 
causing
most indexing programs to falsely identify it as P2.  I cannot say how or why 
the
tNCS test triggers, but the known correct solution in P1 does not contain tNCS.

Ethan


> 
> Then, I went back to the image and processed the datasets with mosflm by 
> checking the indexed spots.
> During this step, I played with the threshold for indexing to follow the 
> strong spot for get correct SG.
> I am not sure whether this is correct or not, but by putting high threshold 
> for indexing (e.g. ~15) I could index the data with C2 which has half 
> dimension for a,b axes (116.348,  67.218, 182.861,  90, 90, 90) to the 
> original unit cell (232.533, 134.202, 182.67, 90, 90, 90).
> With this, I could put 3 molecules in ASU by MR. During refinement, I felt 
> that the R values were not dropping, and I applied twin refinement.
> without twin refinement the R values were (0.39/0.44, work/free), and 
> applying twin refinement gave me significantly better values (0.23/0.26).
> Because there were 6 twin operators which may cause this huge R value drop, I 
> speculate whether this is true or not.
> 
> Your comments will be greatly helpful! 
> 
> With you all the best,
> Donghyuk
> 
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1


-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742



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Re: [ccp4bb] translational NCS & twinning

[Phil Jeffrey]

Donghyuk

The combination of two things gives me cause for concern:
1.  You've reindexed something that apparently scaled OK in point group 
622 into point group 2, with a smaller cell.  Since it's hard to fake 
that sort of data agreement in 622, I assume your data is at the very 
least pseudo-622.
2.  You've modeled that additional symmetry using a whole array of twin 
operators and some non-crystallographic symmetry.


This may in fact be the correct model, but there's a significant risk 
that you're inappropriately modeling something.


Let's assume for a moment that your small-cell C2 refinement with 6 twin 
ops improves the model somewhat.  Now go back and generate scaled data 
sets in all possible point groups suggested by Pointless for that data, 
and try and find molecular replacement solutions with your 
partially-refined model by testing every possible space group in every 
possible point group based on the Pointless suggestions.  The idea is to 
try and model more of the 622 pseudo-symmetry as crystallographic 
symmetry, using fewer twin operators in refinement.  If you test all of 
these combinations, which might be quite extensive, you might find one 
or more that fit nearly as well as your current C2 solution and has 
higher symmetry. You should take a hard look at that those potential 
solutions.  Only when you've thoroughly exhausted alternative molecular 
replacement solutions would you be confident that your C2 model is in 
fact the only reasonable explanation of your data.


But as it stands it is rather atypical and it warrants further investigation

Additional evidence that your C2 cell is the only reasonable model would 
be identifiable electron density differences between each chain in your 
(presumed) multi-chain model.


Cheers
Phil Jeffrey
Princeton

On 1/10/19 11:08 AM, Donghyuk Shin wrote:

Dear Jacob Keller and Vipul,

Thank you both very much for the reply.
Regarding the R-values, I am just wondering whether the huge gap between 
refinements w/ w/o twin operator can be possible even the crystal is not 
twinned?

Best wishes,
Donghyuk



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning

[Herman . Schreuder]

Dear Donghyuk,

Unfortunately, everything is possible when NCS, twinning etc. get into the 
game. I do not have answers, but some questions for you to think about:
- Do you really have 6 twinning operators, or only one operator and are the 
other operators generated by (non)crystallographic symmetry?
- Both your P6322 cell and your C2 cell have angles of 90 90 90. For P6322, the 
last angle should be 120 and for C2, only the second angle is constrained to be 
90. Maybe you should check that not somewhere something went wrong with the 
cell angles.
- How weak are the reflections that got discarded by halving the a- and b-axes? 
Do they have significant intensity, or is it only noise?
- By shrinking the unit cell, you may have created artificial twinning when in 
the large unit cell the molecules are related by "twinning" (N)CS.
- Since you seem to have found a solution with the small unit cell, you could 
see if you could fit this solution in the large unit cell: Process in P1 in the 
large unit cell and use the ensemble (the complete! unit cell of your C2 
solution, as a search model.
- Your current solution maybe correct after all, but I would analyze it very 
critically.

Best,
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Donghyuk 
Shin
Gesendet: Donnerstag, 10. Januar 2019 11:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] translational NCS & twinning

Dear all,

I am having tough time with my Xtal data sets those seem to be twinned or have 
translational NCS, and it will be greatly appreciated if you can give me some 
advices or comments!

Data was initially processed with XDS and scaled with aimless without 
specifying certain space group (SG).
Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates there is 
non-origin peak after patterson analysis. (attached log) And, I could not get 
the proper MR-solution from this data sets.

Because I read that twinning and tNCS cannot be properly distinguished at high 
SG, I went down to subgroup either P32 or P6 assuming that there is twinning 
which make data set seems to have apparently high SG. (procedure was same 
XDS->aimless but I specified the SG to keep them) Then, xtriage still indicates 
there is non-origin peak as before, but found twin laws for the data sets 
(attached log).
However, I still could not get the right MR-solution.
Then, I went even further down to P3 or C2, and xtriage found more twin laws 
which is make sense because of the lower SG. (attached log) Again, I could not 
get the MR-solution.
For all the MR running above, I assumed that phaser(ccp4 module) automatically 
applied tNCS if they present. or should I have to tick on button in the expert 
parameters?

Then, I went back to the image and processed the datasets with mosflm by 
checking the indexed spots.
During this step, I played with the threshold for indexing to follow the strong 
spot for get correct SG.
I am not sure whether this is correct or not, but by putting high threshold for 
indexing (e.g. ~15) I could index the data with C2 which has half dimension for 
a,b axes (116.348,  67.218, 182.861,  90, 90, 90) to the original unit cell 
(232.533, 134.202, 182.67, 90, 90, 90).
With this, I could put 3 molecules in ASU by MR. During refinement, I felt that 
the R values were not dropping, and I applied twin refinement.
without twin refinement the R values were (0.39/0.44, work/free), and applying 
twin refinement gave me significantly better values (0.23/0.26).
Because there were 6 twin operators which may cause this huge R value drop, I 
speculate whether this is true or not.

Your comments will be greatly helpful! 

With you all the best,
Donghyuk





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Re: [ccp4bb] translational NCS & twinning

[Donghyuk Shin]

Dear Jacob Keller and Vipul,

Thank you both very much for the reply.
Regarding the R-values, I am just wondering whether the huge gap between 
refinements w/ w/o twin operator can be possible even the crystal is not 
twinned?

Best wishes,
Donghyuk



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Re: [ccp4bb] translational NCS & twinning

[Keller, Jacob]

>>I feel you went ahead with right strategy.

I agree with this part regarding lowering symmetry.

>>For 2.1 A datasrt, the appropriate drop in Rfree/ Rwork is a strong 
>>indicator, i believe.

This is not true—even non-twinned data will improve in R values with twinning 
operators added as parameters. And twin-refined structures always have lower R 
values.
JPK


On Thu 10 Jan, 2019, 3:52 PM Donghyuk Shin 
mailto:sdh...@gmail.com> wrote:
Dear all,

I am having tough time with my Xtal data sets those seem to be twinned or have 
translational NCS, and it will be greatly appreciated if you can give me some 
advices or comments!

Data was initially processed with XDS and scaled with aimless without 
specifying certain space group (SG).
Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates there is 
non-origin peak after patterson analysis. (attached log)
And, I could not get the proper MR-solution from this data sets.

Because I read that twinning and tNCS cannot be properly distinguished at high 
SG, I went down to subgroup either P32 or P6 assuming that there is twinning 
which make data set seems to have apparently high SG. (procedure was same 
XDS->aimless but I specified the SG to keep them)
Then, xtriage still indicates there is non-origin peak as before, but found 
twin laws for the data sets (attached log).
However, I still could not get the right MR-solution.
Then, I went even further down to P3 or C2, and xtriage found more twin laws 
which is make sense because of the lower SG. (attached log)
Again, I could not get the MR-solution.
For all the MR running above, I assumed that phaser(ccp4 module) automatically 
applied tNCS if they present. or should I have to tick on button in the expert 
parameters?

Then, I went back to the image and processed the datasets with mosflm by 
checking the indexed spots.
During this step, I played with the threshold for indexing to follow the strong 
spot for get correct SG.
I am not sure whether this is correct or not, but by putting high threshold for 
indexing (e.g. ~15) I could index the data with C2 which has half dimension for 
a,b axes (116.348,  67.218, 182.861,  90, 90, 90) to the original unit cell 
(232.533, 134.202, 182.67, 90, 90, 90).
With this, I could put 3 molecules in ASU by MR. During refinement, I felt that 
the R values were not dropping, and I applied twin refinement.
without twin refinement the R values were (0.39/0.44, work/free), and applying 
twin refinement gave me significantly better values (0.23/0.26).
Because there were 6 twin operators which may cause this huge R value drop, I 
speculate whether this is true or not.

Your comments will be greatly helpful!

With you all the best,
Donghyuk





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Re: [ccp4bb] translational NCS & twinning

[vipul panchal]

Hi Donghyuk,
I feel you went ahead with right strategy.
For 2.1 A datasrt, the appropriate drop in Rfree/ Rwork is a strong
indicator, i believe.
If you have already build all possible model, using tls can be of further
help.

Cheers,
Vipul




On Thu 10 Jan, 2019, 3:52 PM Donghyuk Shin  Dear all,
>
> I am having tough time with my Xtal data sets those seem to be twinned or
> have translational NCS, and it will be greatly appreciated if you can give
> me some advices or comments!
>
> Data was initially processed with XDS and scaled with aimless without
> specifying certain space group (SG).
> Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates
> there is non-origin peak after patterson analysis. (attached log)
> And, I could not get the proper MR-solution from this data sets.
>
> Because I read that twinning and tNCS cannot be properly distinguished at
> high SG, I went down to subgroup either P32 or P6 assuming that there is
> twinning which make data set seems to have apparently high SG. (procedure
> was same XDS->aimless but I specified the SG to keep them)
> Then, xtriage still indicates there is non-origin peak as before, but
> found twin laws for the data sets (attached log).
> However, I still could not get the right MR-solution.
> Then, I went even further down to P3 or C2, and xtriage found more twin
> laws which is make sense because of the lower SG. (attached log)
> Again, I could not get the MR-solution.
> For all the MR running above, I assumed that phaser(ccp4 module)
> automatically applied tNCS if they present. or should I have to tick on
> button in the expert parameters?
>
> Then, I went back to the image and processed the datasets with mosflm by
> checking the indexed spots.
> During this step, I played with the threshold for indexing to follow the
> strong spot for get correct SG.
> I am not sure whether this is correct or not, but by putting high
> threshold for indexing (e.g. ~15) I could index the data with C2 which has
> half dimension for a,b axes (116.348,  67.218, 182.861,  90, 90, 90) to the
> original unit cell (232.533, 134.202, 182.67, 90, 90, 90).
> With this, I could put 3 molecules in ASU by MR. During refinement, I felt
> that the R values were not dropping, and I applied twin refinement.
> without twin refinement the R values were (0.39/0.44, work/free), and
> applying twin refinement gave me significantly better values (0.23/0.26).
> Because there were 6 twin operators which may cause this huge R value
> drop, I speculate whether this is true or not.
>
> Your comments will be greatly helpful!
>
> With you all the best,
> Donghyuk
>
>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



To unsubscribe from the CCP4BB list, click the following link:
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[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] Translational NCS with one molecule in ASU

[Herman . Schreuder]

Dear Mark and Remy,

This is what I meant by statistical disorder. It goes back to a rather cryptic 
remark by Zbyszek Otwinowski (Jacob, you should have googled Otwinowski instead 
of Ajees  ;-)  !) that there is a twinning continuum with sharp spots at both 
extremes and streaky spots in between. However, since his explanation is 
difficult to understand for mere mortal crystallographers like myself, I will 
describe below how I see it (which may not be entirely mathematically correct):

1) large twin domains: This is classical twinning. One sees diffraction of two 
different crystals (domains) which happen to be perfectly aligned. The crystal 
domains are not within coherent range and I's are added instead of F's and the 
spots are perfectly sharp.
2) intermediate size twin domains: I suspect the twin domains will be around 10 
unit cells, but this might be completely off. Here we see streaky spots. An 
explanation I read somewhere is that this is due to the small domain size. In a 
diffraction experiment: with two slits one gets very broad maxima, with say 10 
slits the maxima are already sharper and with 100 slits, one gets very sharp 
maxima. So in this case the domains are too small to generate sharp spots.
3) statistical disorder: Here one can no longer speak from twin domains, the 
unit cells (molecules?) are randomly distributed in the crystal and the crystal 
is "homogeneous" again resulting in sharp spots. This is what I think is the 
case here.

Just a 0.5 lattice translocation where the complete layer is translated will 
not produce the observed Patterson peak, since the intermolecular vectors are 
just translated and do not change. However, if a 2-fold axis is randomly 
skipped, two molecules which should be antiparallel are suddenly parallel and 
this will produce the observed Patterson peak. It could also explain the 
absence of alternating strong and weak reflections in the data set.

As I said, it will be a very nice puzzle to solve!

Herman



-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Mark 
Wilson
Gesendet: Donnerstag, 3. September 2015 19:56
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] AW: [ccp4bb] Translational NCS with one molecule in ASU

Dear Remy,
Indeed, I think you may be correct and we're pursuing this now.  A perfect
0.5 lattice translocation along b in P212121 would (I think) result in the 
pathology we observe.  We do not see zones of streaked reflections in the 
images, but my thinking is that if the lattice defect is coincident with a 
crystallographic translation operator, perhaps we wouldn't expect to.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 9/3/15 12:49 PM, "Remy Loris" <relo...@vub.ac.be> wrote:

>Dear Mark,
>
>My suspicion is that what you observe here is a lattice disorder, 
>possibly related to what is described in
>
>Jimin Wang, Satwik Kamtekar, Andrea J. Berman and Thomas A. Steitz
>(2005) Correction of X-ray intensities from single crystals containing 
>lattice-translocation defects Acta Cryst D61,  67-74.
>
>If your unit cell is offset statistically by 0.5 in the b-direction, 
>this should provide such a strong non-origin peak as you observe. In 
>the cases that have been described until now, this type of disorder 
>also involves zones of nice sharp reflections and other zones with more 
>streaky reflections. Do you see something similar?
>In order to use such data, they have to be corrected as described in 
>the paper above.
>
>Possibly, the crystals with the 10% non-origin peak also have the 
>disorder, but much less pronounced so that omitting the required 
>correction did not prevent structure determination and refinement 
>(similar to let say a small fraction of merohedral twinning that is 
>overlooked).
>
>Remy Loris
>Vrije Universiteit Brussel and VIB
>
>DOI: 10.1107/S0907444904026721
>
>On 03/09/15 18:13, George Sheldrick wrote:
>> Dear Mark,
>>
>> Since your resolution is good enough, perhaps you should try to solve 
>> it ab initio with Arcimboldo Lite. This has already solved a number 
>> of structures that turned out to be unexpected.
>>
>> Best wishes, George
>>
>>
>> On 09/03/2015 05:44 PM, Mark Wilson wrote:
>>> Hi Herman,
>>> A fair point-the odds of "depressing coincidence" do seem to be  
>>>climbing!
>>> We did inspect the deposited data for a similar peak and, while one 
>>>is  present, it is only ~10% of the origin and at a different location.
>>> We'll
>>> do some due diligence on our end by re-dissolving crystals and  
>>>performing  ma

[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] Translational NCS with one molecule in ASU

[Herman . Schreuder]

It just occurred to me that a 0.5 lattice translocation will bring molecules 
from neighboring layers parallel to each other, which would also explain the 
observed patterson peak.

Sorry,
Herman

-Ursprüngliche Nachricht-
Von: Schreuder, Herman R/DE 
Gesendet: Freitag, 4. September 2015 10:06
An: CCP4BB@JISCMAIL.AC.UK
Betreff: AW: [ccp4bb] AW: [ccp4bb] Translational NCS with one molecule in ASU

Dear Mark and Remy,

This is what I meant by statistical disorder. It goes back to a rather cryptic 
remark by Zbyszek Otwinowski (Jacob, you should have googled Otwinowski instead 
of Ajees  ;-)  !) that there is a twinning continuum with sharp spots at both 
extremes and streaky spots in between. However, since his explanation is 
difficult to understand for mere mortal crystallographers like myself, I will 
describe below how I see it (which may not be entirely mathematically correct):

1) large twin domains: This is classical twinning. One sees diffraction of two 
different crystals (domains) which happen to be perfectly aligned. The crystal 
domains are not within coherent range and I's are added instead of F's and the 
spots are perfectly sharp.
2) intermediate size twin domains: I suspect the twin domains will be around 10 
unit cells, but this might be completely off. Here we see streaky spots. An 
explanation I read somewhere is that this is due to the small domain size. In a 
diffraction experiment: with two slits one gets very broad maxima, with say 10 
slits the maxima are already sharper and with 100 slits, one gets very sharp 
maxima. So in this case the domains are too small to generate sharp spots.
3) statistical disorder: Here one can no longer speak from twin domains, the 
unit cells (molecules?) are randomly distributed in the crystal and the crystal 
is "homogeneous" again resulting in sharp spots. This is what I think is the 
case here.

Just a 0.5 lattice translocation where the complete layer is translated will 
not produce the observed Patterson peak, since the intermolecular vectors are 
just translated and do not change. However, if a 2-fold axis is randomly 
skipped, two molecules which should be antiparallel are suddenly parallel and 
this will produce the observed Patterson peak. It could also explain the 
absence of alternating strong and weak reflections in the data set.

As I said, it will be a very nice puzzle to solve!

Herman



-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Mark 
Wilson
Gesendet: Donnerstag, 3. September 2015 19:56
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] AW: [ccp4bb] Translational NCS with one molecule in ASU

Dear Remy,
Indeed, I think you may be correct and we're pursuing this now.  A perfect
0.5 lattice translocation along b in P212121 would (I think) result in the 
pathology we observe.  We do not see zones of streaked reflections in the 
images, but my thinking is that if the lattice defect is coincident with a 
crystallographic translation operator, perhaps we wouldn't expect to.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 9/3/15 12:49 PM, "Remy Loris" <relo...@vub.ac.be> wrote:

>Dear Mark,
>
>My suspicion is that what you observe here is a lattice disorder, 
>possibly related to what is described in
>
>Jimin Wang, Satwik Kamtekar, Andrea J. Berman and Thomas A. Steitz
>(2005) Correction of X-ray intensities from single crystals containing 
>lattice-translocation defects Acta Cryst D61,  67-74.
>
>If your unit cell is offset statistically by 0.5 in the b-direction, 
>this should provide such a strong non-origin peak as you observe. In 
>the cases that have been described until now, this type of disorder 
>also involves zones of nice sharp reflections and other zones with more 
>streaky reflections. Do you see something similar?
>In order to use such data, they have to be corrected as described in 
>the paper above.
>
>Possibly, the crystals with the 10% non-origin peak also have the 
>disorder, but much less pronounced so that omitting the required 
>correction did not prevent structure determination and refinement 
>(similar to let say a small fraction of merohedral twinning that is 
>overlooked).
>
>Remy Loris
>Vrije Universiteit Brussel and VIB
>
>DOI: 10.1107/S0907444904026721
>
>On 03/09/15 18:13, George Sheldrick wrote:
>> Dear Mark,
>>
>> Since your resolution is good enough, perhaps you should try to solve 
>> it ab initio with Arcimboldo Lite. This has already solved a number 
>> of structures that turned out to be unexpected.
>>
>> Best wishes, George
>>
>>
>> On 09/03/2015 05:44 PM, Mark Wilson wrote:
>>> Hi Herman,

Re: [ccp4bb] AW: [ccp4bb] Translational NCS with one molecule in ASU

[Mark Wilson]

Hi Herman,
A fair point-the odds of "depressing coincidence" do seem to be climbing!
We did inspect the deposited data for a similar peak and, while one is
present, it is only ~10% of the origin and at a different location.  We'll
do some due diligence on our end by re-dissolving crystals and performing
mass spec.  As there seems to be some interest in this, I'll update once
we've figured it out, even if it's an embarrassing case of wrong protein,
same cell.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 9/3/15 10:25 AM, "CCP4 bulletin board on behalf of
herman.schreu...@sanofi.com" <CCP4BB@JISCMAIL.AC.UK on behalf of
herman.schreu...@sanofi.com> wrote:

>Dear Mark,
>
>In this case you will have to apply Baysian statistics: given the prior:
>same protein, same space group same cell dimensions and molecular
>replacement fails completely, the likelihood of having some depressing
>coincidence somewhere is approaches 100%!
>
>What I would do in addition to excellent suggestions you already got, is
>to try to download the Fobs from the pdb for the structures with the same
>protein, space group and cell dimensions, and calculate pattersons with
>those. Sometimes strong peaks appear in pattersons for no obvious reasons.
>I would also consider statistical disorder, which will not show up in
>twinning statistics since in this case F's (including phases) are added
>instead of I's. Anyways, it will be an interesting puzzle to solve!
>
>Good luck,
>Herman
>
>
>-Ursprüngliche Nachricht-
>Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von
>Mark Wilson
>Gesendet: Donnerstag, 3. September 2015 02:06
>An: CCP4BB@JISCMAIL.AC.UK
>Betreff: Re: [ccp4bb] Translational NCS with one molecule in ASU
>
>Dear CCP4 Community,
>I've had a number of helpful responses (on- and off-list) that I will
>briefly summarize via response, including information that I probably
>should have included in the original post.  Many have suggested a wrong
>space group, which I agree seems probable.  MR was attempted in PHASER
>with all possible choices of space group for a primitive orthorhombic
>lattice, and in all cases failed with no rotation or translation peaks
>above a Z-score of 5.
>   I've not yet tried monoclinic lattices and will, but this still wouldn't
>explain (to me anyway) an apparently impossible combination of
>translational NCS in P212121 with a cell that can't accommodate a second
>molecule unless twinning was also present, which may be the case (as
>Eleanor suggested).  Others have asked about evidence of missed weak
>reflections indicating a larger true cell, which I looked for but didn't
>see in these images.  The crystal that was used was mounted at room
>temperature, so there is no opportunity for cryo artifacts to have done
>something strange to the cell.
>   Other suggestions included the presence of strong internal symmetry in
>the molecule, which is present, but as a pseudo-threefold, which seems
>incompatible with my NCS centering operation.  One respondent suggested
>that we've crystallized the wrong molecule, which is something I also
>worried about a bit.  Although possible, the space group and cell for our
>crystal are both as previously reported for this protein by another
>group, so it would be a depressing coincidence if we crystallized the
>wrong protein in the same cell. I'll be happy to update if/when we figure
>this out should it be of interest to the board. Thank you all for your
>thoughtful responses, which arrived in impressive number in the time it
>took me to drive home.
>Best regards,
>Mark
>
>Mark A. Wilson
>Associate Professor
>Department of Biochemistry/Redox Biology Center University of Nebraska
>N118 Beadle Center
>1901 Vine Street
>Lincoln, NE 68588
>(402) 472-3626
>mwilso...@unl.edu 
>
>
>
>
>
>On 9/2/15 5:30 PM, "CCP4 bulletin board on behalf of Eleanor Dodson"
><CCP4BB@JISCMAIL.AC.UK on behalf of eleanor.dod...@york.ac.uk> wrote:
>
>>Well -  a translation of 0 0.5 0 would generate absences along b so
>>that the SG could be P212121 or P 21 2 21Š
>>
>>
>>I would suspect twinning and a monoclinic SG .
>>Or as we found sadly - half the protein had disappeared in the
>>crystallisation trials..
>>
>>
>>But such a translation must mean you almost have a halved unit cell?
>>Another way of saying there isn't enough room for your molecule..
>>
>>
>>
>>On 2 September 2015 at 22:38, Shane Caldwell
>><shane.caldwel...@gmail.com> wrote:
>>

Re: [ccp4bb] Translational NCS with one molecule in ASU

[Mark Wilson]

Hi All,
An important point is that the cell dimensions are: 54.98 58.45 66.89 90
90 90.  While a and b are similar, they are (in my opinion) not similar
enough to support pseudo-merohedral twinning.  The absences are indeed
absent, with I/sigma(I) between ~ -1 and 2.  All three axes have similarly
convincing absences in P212121, included below.
Best regards,
Mark
==

 Intensities of systematic absences
  h   k   l  Intensity Sigma   I/Sigma

  0   0   5   1.8   2.5   0.7
  0   0   7  -4.1   3.5  -1.2
  0   0   9   9.9   4.6   2.2
  0   0  11  -1.1   6.5  -0.2
  0   0  13  12.9  10.0   1.3
  0   0  15  -7.2   9.9  -0.7
  0   0  17 -11.2   8.8  -1.3
  0   0  19   1.1  10.0   0.1
  0   0  21  -1.4   9.3  -0.2
  0   0  23   0.8   9.3   0.1
  0   0  25   1.5   8.1   0.2
  0   0  27   7.4   9.3   0.8
  0   3   0  -1.6   4.5  -0.4
  0   5   0  -4.7   5.5  -0.9
  0   7   0 -18.1  12.2  -1.5
  0   9   0  13.0  13.6   1.0
  0  11   0  19.3  18.6   1.0
  0  13   0  51.6  29.8   1.7
  0  15   0  38.6  24.4   1.6
  0  17   0  -9.5  30.2  -0.3
  0  19   0 -42.8  36.1  -1.2
  0  21   0 -12.5  22.4  -0.6
  0  23   0  -3.2  24.9  -0.1
  0  25   0  54.8  38.7   1.4
  0  27   0 -28.2  36.2  -0.8
  0  29   0  -0.1  30.6   0.0
  0  31   0 -25.7  39.8  -0.6
  0  33   0 -11.6  38.1  -0.3
  3   0   0   1.6   3.3   0.5
  5   0   0   9.2   5.2   1.8
  7   0   0  -0.4   9.6   0.0
  9   0   0  -2.7  11.2  -0.2
 11   0   0  65.1  21.1   3.1
 13   0   0  19.8  19.0   1.0
 15   0   0 -24.5  20.9  -1.2
 17   0   0  29.9  31.8   0.9
 19   0   0  -3.3  24.3  -0.1
 21   0   0 -33.4  33.0  -1.0
 23   0   0  -4.8  29.4  -0.2
 25   0   0  -6.7  33.3  -0.2
 27   0   0  55.3  37.6   1.5
 29   0   0  11.0  38.6   0.3
 31   0   0 -36.4  46.6  -0.8




Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 9/3/15 1:51 AM, "CCP4 bulletin board on behalf of Adrian Goldman"
 wrote:

>I agree. This was essentially Eleanor's viewpoint too.  So p21
>merohedrally twinned to orthorhombic with the 0.47 translational ncs.. I
>would look at the h00, k00, l00 odd peaks (the ones systematically
>disallowed) to look for evidence of some intensity
> along one of the axes in the disallowed spots.  That will help establish
>which axis has the true 21 screw. My guess is that at high Bragg angle
>there is some intensity in the k odd spots at least.
>
>
>Adrian
>
>Sent from my iPad
>
>On 3 Sep 2015, at 6:40 am, Sudipta Bhattacharyya
> wrote:
>
>
>
>Hi Mark,
>
>
>One strategy that worked for me is to reprocess/expand the data to P1 and
>try to do MR in that SG, do initial one cycle of rigid body and
>restrained refinements and then you can feed the P1 data and the model to
>zanuda to get correct assessment of SG.
> Then you have to reprocess the data accordingly. Anyway, twining tests
>are most of the time obscured when tNCS is present. So, I think, the
>presence of twining can't be simply overruled in this case. reprocessing
>the data in monoclinic SG, followed by doing
> MR could be the key to this problem.
>
>
>Good luck..!!!
>Sudipta  
>
>
>On Wed, Sep 2, 2015 at 6:05 PM, Mark Wilson
> wrote:
>
>Dear CCP4 Community,
>I've had a number of helpful responses (on- and off-list) that I will
>briefly summarize via response, including information that I probably
>should have included in the original post.  Many have suggested a wrong
>space group, which I agree seems probable.  MR was attempted in PHASER
>with all possible choices of space group for a primitive orthorhombic
>lattice, and in all cases failed with no rotation or translation peaks
>above a Z-score of 5.
>I've not yet tried monoclinic lattices and will, but this still
>wouldn't
>explain (to me anyway) an apparently impossible combination of
>translational NCS in P212121 with a cell that can't accommodate a second
>molecule unless twinning was also present, which may be the case (as
>Eleanor suggested).  Others have asked about evidence of missed weak
>reflections indicating a larger true cell, which I looked for but didn't
>see in these 

Re: [ccp4bb] AW: [ccp4bb] Translational NCS with one molecule in ASU

[Mark Wilson]

Dear Remy,
Indeed, I think you may be correct and we're pursuing this now.  A perfect
0.5 lattice translocation along b in P212121 would (I think) result in the
pathology we observe.  We do not see zones of streaked reflections in the
images, but my thinking is that if the lattice defect is coincident with a
crystallographic translation operator, perhaps we wouldn't expect to.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 9/3/15 12:49 PM, "Remy Loris" <relo...@vub.ac.be> wrote:

>Dear Mark,
>
>My suspicion is that what you observe here is a lattice disorder,
>possibly related to what is described in
>
>Jimin Wang, Satwik Kamtekar, Andrea J. Berman and Thomas A. Steitz
>(2005) Correction of X-ray intensities from single crystals containing
>lattice-translocation defects Acta Cryst D61,  67-74.
>
>If your unit cell is offset statistically by 0.5 in the b-direction,
>this should provide such a strong non-origin peak as you observe. In the
>cases that have been described until now, this type of disorder also
>involves zones of nice sharp reflections and other zones with more
>streaky reflections. Do you see something similar?
>In order to use such data, they have to be corrected as described in the
>paper above.
>
>Possibly, the crystals with the 10% non-origin peak also have the
>disorder, but much less pronounced so that omitting the required
>correction did not prevent structure determination and refinement
>(similar to let say a small fraction of merohedral twinning that is
>overlooked).
>
>Remy Loris
>Vrije Universiteit Brussel and VIB
>
>DOI: 10.1107/S0907444904026721
>
>On 03/09/15 18:13, George Sheldrick wrote:
>> Dear Mark,
>>
>> Since your resolution is good enough, perhaps you should try to solve
>> it ab initio with Arcimboldo Lite. This has already solved a number of
>> structures that turned out to be unexpected.
>>
>> Best wishes, George
>>
>>
>> On 09/03/2015 05:44 PM, Mark Wilson wrote:
>>> Hi Herman,
>>> A fair point-the odds of "depressing coincidence" do seem to be
>>> climbing!
>>> We did inspect the deposited data for a similar peak and, while one is
>>> present, it is only ~10% of the origin and at a different location.
>>> We'll
>>> do some due diligence on our end by re-dissolving crystals and
>>> performing
>>> mass spec.  As there seems to be some interest in this, I'll update
>>>once
>>> we've figured it out, even if it's an embarrassing case of wrong
>>> protein,
>>> same cell.
>>> Best regards,
>>> Mark
>>>
>>> Mark A. Wilson
>>> Associate Professor
>>> Department of Biochemistry/Redox Biology Center
>>> University of Nebraska
>>> N118 Beadle Center
>>> 1901 Vine Street
>>> Lincoln, NE 68588
>>> (402) 472-3626
>>> mwilso...@unl.edu
>>>
>>>
>>>
>>>
>>>
>>>
>>> On 9/3/15 10:25 AM, "CCP4 bulletin board on behalf of
>>> herman.schreu...@sanofi.com"<CCP4BB@JISCMAIL.AC.UK on behalf of
>>> herman.schreu...@sanofi.com>  wrote:
>>>
>>>> Dear Mark,
>>>>
>>>> In this case you will have to apply Baysian statistics: given the
>>>> prior:
>>>> same protein, same space group same cell dimensions and molecular
>>>> replacement fails completely, the likelihood of having some depressing
>>>> coincidence somewhere is approaches 100%!
>>>>
>>>> What I would do in addition to excellent suggestions you already
>>>> got, is
>>>> to try to download the Fobs from the pdb for the structures with the
>>>> same
>>>> protein, space group and cell dimensions, and calculate pattersons
>>>>with
>>>> those. Sometimes strong peaks appear in pattersons for no obvious
>>>> reasons.
>>>> I would also consider statistical disorder, which will not show up in
>>>> twinning statistics since in this case F's (including phases) are
>>>>added
>>>> instead of I's. Anyways, it will be an interesting puzzle to solve!
>>>>
>>>> Good luck,
>>>> Herman
>>>>
>>>>
>>>> -Ursprüngliche Nachricht-
>>>> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von
>>>> Mark Wilson
>>>> Gesendet: Donnerstag, 

Re: [ccp4bb] AW: [ccp4bb] Translational NCS with one molecule in ASU

[Mark Wilson]

>>>> Associate Professor
>>>> Department of Biochemistry/Redox Biology Center
>>>> University of Nebraska
>>>> N118 Beadle Center
>>>> 1901 Vine Street
>>>> Lincoln, NE 68588
>>>> (402) 472-3626
>>>> mwilso...@unl.edu
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>> On 9/3/15 10:25 AM, "CCP4 bulletin board on behalf of
>>>> herman.schreu...@sanofi.com"<CCP4BB@JISCMAIL.AC.UK on behalf of
>>>> herman.schreu...@sanofi.com>  wrote:
>>>>
>>>>> Dear Mark,
>>>>>
>>>>> In this case you will have to apply Baysian statistics: given the
>>>>> prior:
>>>>> same protein, same space group same cell dimensions and molecular
>>>>> replacement fails completely, the likelihood of having some
>>>>>depressing
>>>>> coincidence somewhere is approaches 100%!
>>>>>
>>>>> What I would do in addition to excellent suggestions you already
>>>>> got, is
>>>>> to try to download the Fobs from the pdb for the structures with the
>>>>> same
>>>>> protein, space group and cell dimensions, and calculate pattersons
>>>>>with
>>>>> those. Sometimes strong peaks appear in pattersons for no obvious
>>>>> reasons.
>>>>> I would also consider statistical disorder, which will not show up in
>>>>> twinning statistics since in this case F's (including phases) are
>>>>>added
>>>>> instead of I's. Anyways, it will be an interesting puzzle to solve!
>>>>>
>>>>> Good luck,
>>>>> Herman
>>>>>
>>>>>
>>>>> -Ursprüngliche Nachricht-
>>>>> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag
>>>>>von
>>>>> Mark Wilson
>>>>> Gesendet: Donnerstag, 3. September 2015 02:06
>>>>> An: CCP4BB@JISCMAIL.AC.UK
>>>>> Betreff: Re: [ccp4bb] Translational NCS with one molecule in ASU
>>>>>
>>>>> Dear CCP4 Community,
>>>>> I've had a number of helpful responses (on- and off-list) that I will
>>>>> briefly summarize via response, including information that I probably
>>>>> should have included in the original post.  Many have suggested a
>>>>>wrong
>>>>> space group, which I agree seems probable.  MR was attempted in
>>>>>PHASER
>>>>> with all possible choices of space group for a primitive orthorhombic
>>>>> lattice, and in all cases failed with no rotation or translation
>>>>>peaks
>>>>> above a Z-score of 5.
>>>>> I've not yet tried monoclinic lattices and will, but this still
>>>>> wouldn't
>>>>> explain (to me anyway) an apparently impossible combination of
>>>>> translational NCS in P212121 with a cell that can't accommodate a
>>>>> second
>>>>> molecule unless twinning was also present, which may be the case (as
>>>>> Eleanor suggested).  Others have asked about evidence of missed weak
>>>>> reflections indicating a larger true cell, which I looked for but
>>>>> didn't
>>>>> see in these images.  The crystal that was used was mounted at room
>>>>> temperature, so there is no opportunity for cryo artifacts to have
>>>>>done
>>>>> something strange to the cell.
>>>>> Other suggestions included the presence of strong internal
>>>>> symmetry in
>>>>> the molecule, which is present, but as a pseudo-threefold, which
>>>>>seems
>>>>> incompatible with my NCS centering operation.  One respondent
>>>>>suggested
>>>>> that we've crystallized the wrong molecule, which is something I also
>>>>> worried about a bit.  Although possible, the space group and cell
>>>>> for our
>>>>> crystal are both as previously reported for this protein by another
>>>>> group, so it would be a depressing coincidence if we crystallized the
>>>>> wrong protein in the same cell. I'll be happy to update if/when we
>>>>> figure
>>>>> this out should it be of interest to the board. Thank you all for
>>>>>your
>>>>> thoughtful responses, which arrived in impressive

Re: [ccp4bb] AW: [ccp4bb] Translational NCS with one molecule in ASU

[Mark Wilson]

>>>> this should provide such a strong non-origin peak as you observe. In
>>>>the
>>>> cases that have been described until now, this type of disorder also
>>>> involves zones of nice sharp reflections and other zones with more
>>>> streaky reflections. Do you see something similar?
>>>> In order to use such data, they have to be corrected as described in
>>>>the
>>>> paper above.
>>>>
>>>> Possibly, the crystals with the 10% non-origin peak also have the
>>>> disorder, but much less pronounced so that omitting the required
>>>> correction did not prevent structure determination and refinement
>>>> (similar to let say a small fraction of merohedral twinning that is
>>>> overlooked).
>>>>
>>>> Remy Loris
>>>> Vrije Universiteit Brussel and VIB
>>>>
>>>> DOI: 10.1107/S0907444904026721
>>>>
>>>>> On 03/09/15 18:13, George Sheldrick wrote:
>>>>> Dear Mark,
>>>>>
>>>>> Since your resolution is good enough, perhaps you should try to solve
>>>>> it ab initio with Arcimboldo Lite. This has already solved a number
>>>>>of
>>>>> structures that turned out to be unexpected.
>>>>>
>>>>> Best wishes, George
>>>>>
>>>>>
>>>>>> On 09/03/2015 05:44 PM, Mark Wilson wrote:
>>>>>> Hi Herman,
>>>>>> A fair point-the odds of "depressing coincidence" do seem to be
>>>>>> climbing!
>>>>>> We did inspect the deposited data for a similar peak and, while one
>>>>>>is
>>>>>> present, it is only ~10% of the origin and at a different location.
>>>>>> We'll
>>>>>> do some due diligence on our end by re-dissolving crystals and
>>>>>> performing
>>>>>> mass spec.  As there seems to be some interest in this, I'll update
>>>>>> once
>>>>>> we've figured it out, even if it's an embarrassing case of wrong
>>>>>> protein,
>>>>>> same cell.
>>>>>> Best regards,
>>>>>> Mark
>>>>>>
>>>>>> Mark A. Wilson
>>>>>> Associate Professor
>>>>>> Department of Biochemistry/Redox Biology Center
>>>>>> University of Nebraska
>>>>>> N118 Beadle Center
>>>>>> 1901 Vine Street
>>>>>> Lincoln, NE 68588
>>>>>> (402) 472-3626
>>>>>> mwilso...@unl.edu
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> On 9/3/15 10:25 AM, "CCP4 bulletin board on behalf of
>>>>>> herman.schreu...@sanofi.com"<CCP4BB@JISCMAIL.AC.UK on behalf of
>>>>>> herman.schreu...@sanofi.com>  wrote:
>>>>>>
>>>>>>> Dear Mark,
>>>>>>>
>>>>>>> In this case you will have to apply Baysian statistics: given the
>>>>>>> prior:
>>>>>>> same protein, same space group same cell dimensions and molecular
>>>>>>> replacement fails completely, the likelihood of having some
>>>>>>> depressing
>>>>>>> coincidence somewhere is approaches 100%!
>>>>>>>
>>>>>>> What I would do in addition to excellent suggestions you already
>>>>>>> got, is
>>>>>>> to try to download the Fobs from the pdb for the structures with
>>>>>>>the
>>>>>>> same
>>>>>>> protein, space group and cell dimensions, and calculate pattersons
>>>>>>> with
>>>>>>> those. Sometimes strong peaks appear in pattersons for no obvious
>>>>>>> reasons.
>>>>>>> I would also consider statistical disorder, which will not show up
>>>>>>>in
>>>>>>> twinning statistics since in this case F's (including phases) are
>>>>>>> added
>>>>>>> instead of I's. Anyways, it will be an interesting puzzle to solve!
>>>>>>>
>>>>>>> Good luck,
>>>>>>> Herman
>>>>>>>
>>>>>>>
>>>>>>> -Ursprüngliche Nachri

Re: [ccp4bb] Translational NCS and molecular replacement.

[Sudipta Bhattacharyya]

Dear community,

Thanks for your valuable suggestions.

Best regards,
Sudipta.


On Thu, Jul 17, 2014 at 8:04 AM, Eleanor Dodson eleanor.dod...@york.ac.uk
wrote:

 No need to reindex - just do this to change the space group in the scala
 header.
 mtzutils hklin1 scala.mtz hklout scala-P22121.mtz
 symm P22121
 end

 There are other ways of course..
 Eleanor



 On 16 July 2014 13:28, Vajdos, Felix felix.vaj...@pfizer.com wrote:

  Dear Sudipta—



 Herman is correct, you just need to reindex your scala.mtz file to the
 correct space group.



 Regarding translational NCS, it has been many years since I’ve dealt with
 this, but it is my impression that modern refinement methods treat this
 much better, especially with maximum likelihood targets, as the low(er)
 intensity reflections will have systematically lower sig/noise than the
 other parity groups.



 *Felix F. Vajdos*

 * Associate Research Fellow Structural Biology  Biophysics*
 *Pfizer Inc*
 *Eastern Point Road *
 *MS 8220-3273*
 *Groton, CT  06334*

 *860-715-6504 860-715-6504*







 *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
 *herman.schreu...@sanofi.com
 *Sent:* Wednesday, July 16, 2014 3:08 AM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* [ccp4bb] AW: [ccp4bb] Translational NCS and molecular
 replacement.



 Dear Sudipta,

 you are correct, your original scala.mtz has the wrong space group in it,
 resulting in very high Rfactors (and presumably bad electron density).

 In these cases, I usually reprocess (remerge) the data in the correct
 space group to get the statistics right (and gain probably a few extra h00
 reflections that got rejected in P212121). If you use the same a, b and c
 axes as before, you do not need to rerun Phaser, otherwise you have to.

 If you have translational NCS, you have to live with it. The only way to
 get rid of it is to find another crystal with a different crystal packing.



 Best,

 Herman



 *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK
 CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Sudipta Bhattacharyya
 *Gesendet:* Dienstag, 15. Juli 2014 20:32
 *An:* CCP4BB@JISCMAIL.AC.UK
 *Betreff:* [ccp4bb] Translational NCS and molecular replacement.



 Dear Community,



 I have some doubts to clarify. In a way to solve a structure by
 Phaser-MR, I found phaser ended up with a potentially good MR solution
 (with good statistics, packing and electron density, and as we know the
 homolog structure so in a reasonable biological assembly also). However,
 the space group where phaser found the potential solution (P22121) is
 different what we got in pointless (P212121), and phaser also indicated the
 presence of translational NCS (NCS translation vector = 0.500, 0.494
 0.391). Now when we try to refine the structure with its original scala.mtz
 file (which is indexed and sclaled in P212121) and phaser generated pdb
 file, the R/Rfree is very high (around 0.5) but when I tried refining with
 mtz file generated by phaser, it was reasonable, at least for first cycle
 of refinement (R/Rfree, 0.41/0.46). Now my question is, can I use the
 phaser generated mtz file instead of the original scala.mtz for further
 refinement? Or I have to reindex my original data into phaser suggested
 spacegroup and run the MR again? Translational NCS are generally associated
 with high R values, is there any way to get rid of that problem?



 Best regards,

 Sudipta.

Re: [ccp4bb] Translational NCS and molecular replacement.

[Eleanor Dodson]

No need to reindex - just do this to change the space group in the scala
header.
mtzutils hklin1 scala.mtz hklout scala-P22121.mtz
symm P22121
end

There are other ways of course..
Eleanor



On 16 July 2014 13:28, Vajdos, Felix felix.vaj...@pfizer.com wrote:

  Dear Sudipta—



 Herman is correct, you just need to reindex your scala.mtz file to the
 correct space group.



 Regarding translational NCS, it has been many years since I’ve dealt with
 this, but it is my impression that modern refinement methods treat this
 much better, especially with maximum likelihood targets, as the low(er)
 intensity reflections will have systematically lower sig/noise than the
 other parity groups.



 *Felix F. Vajdos*

 * Associate Research Fellow Structural Biology  Biophysics*
 *Pfizer Inc*
 *Eastern Point Road *
 *MS 8220-3273*
 *Groton, CT  06334*

 *860-715-6504 860-715-6504*







 *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *
 herman.schreu...@sanofi.com
 *Sent:* Wednesday, July 16, 2014 3:08 AM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* [ccp4bb] AW: [ccp4bb] Translational NCS and molecular
 replacement.



 Dear Sudipta,

 you are correct, your original scala.mtz has the wrong space group in it,
 resulting in very high Rfactors (and presumably bad electron density).

 In these cases, I usually reprocess (remerge) the data in the correct
 space group to get the statistics right (and gain probably a few extra h00
 reflections that got rejected in P212121). If you use the same a, b and c
 axes as before, you do not need to rerun Phaser, otherwise you have to.

 If you have translational NCS, you have to live with it. The only way to
 get rid of it is to find another crystal with a different crystal packing.



 Best,

 Herman



 *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK
 CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Sudipta Bhattacharyya
 *Gesendet:* Dienstag, 15. Juli 2014 20:32
 *An:* CCP4BB@JISCMAIL.AC.UK
 *Betreff:* [ccp4bb] Translational NCS and molecular replacement.



 Dear Community,



 I have some doubts to clarify. In a way to solve a structure by Phaser-MR,
 I found phaser ended up with a potentially good MR solution (with good
 statistics, packing and electron density, and as we know the homolog
 structure so in a reasonable biological assembly also). However, the space
 group where phaser found the potential solution (P22121) is different what
 we got in pointless (P212121), and phaser also indicated the presence of
 translational NCS (NCS translation vector = 0.500, 0.494 0.391). Now when
 we try to refine the structure with its original scala.mtz file (which is
 indexed and sclaled in P212121) and phaser generated pdb file, the R/Rfree
 is very high (around 0.5) but when I tried refining with mtz file generated
 by phaser, it was reasonable, at least for first cycle of refinement
 (R/Rfree, 0.41/0.46). Now my question is, can I use the phaser generated
 mtz file instead of the original scala.mtz for further refinement? Or I
 have to reindex my original data into phaser suggested spacegroup and run
 the MR again? Translational NCS are generally associated with high R
 values, is there any way to get rid of that problem?



 Best regards,

 Sudipta.

[ccp4bb] AW: [ccp4bb] Translational NCS and molecular replacement.

[Herman . Schreuder]

Dear Sudipta,
you are correct, your original scala.mtz has the wrong space group in it, 
resulting in very high Rfactors (and presumably bad electron density).
In these cases, I usually reprocess (remerge) the data in the correct space 
group to get the statistics right (and gain probably a few extra h00 
reflections that got rejected in P212121). If you use the same a, b and c axes 
as before, you do not need to rerun Phaser, otherwise you have to.
If you have translational NCS, you have to live with it. The only way to get 
rid of it is to find another crystal with a different crystal packing.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Sudipta 
Bhattacharyya
Gesendet: Dienstag, 15. Juli 2014 20:32
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Translational NCS and molecular replacement.

Dear Community,

I have some doubts to clarify. In a way to solve a structure by Phaser-MR, I 
found phaser ended up with a potentially good MR solution (with good 
statistics, packing and electron density, and as we know the homolog structure 
so in a reasonable biological assembly also). However, the space group where 
phaser found the potential solution (P22121) is different what we got in 
pointless (P212121), and phaser also indicated the presence of translational 
NCS (NCS translation vector = 0.500, 0.494 0.391). Now when we try to refine 
the structure with its original scala.mtz file (which is indexed and sclaled in 
P212121) and phaser generated pdb file, the R/Rfree is very high (around 0.5) 
but when I tried refining with mtz file generated by phaser, it was reasonable, 
at least for first cycle of refinement (R/Rfree, 0.41/0.46). Now my question 
is, can I use the phaser generated mtz file instead of the original scala.mtz 
for further refinement? Or I have to reindex my original data into phaser 
suggested spacegroup and run the MR again? Translational NCS are generally 
associated with high R values, is there any way to get rid of that problem?

Best regards,
Sudipta.

Re: [ccp4bb] Translational NCS and molecular replacement.

[Vajdos, Felix]

Dear Sudipta—

Herman is correct, you just need to reindex your scala.mtz file to the correct 
space group.

Regarding translational NCS, it has been many years since I’ve dealt with this, 
but it is my impression that modern refinement methods treat this much better, 
especially with maximum likelihood targets, as the low(er) intensity 
reflections will have systematically lower sig/noise than the other parity 
groups.

Felix F. Vajdos
Associate Research Fellow
Structural Biology  Biophysics
Pfizer Inc
Eastern Point Road
MS 8220-3273
Groton, CT  06334
860-715-6504



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
herman.schreu...@sanofi.com
Sent: Wednesday, July 16, 2014 3:08 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] Translational NCS and molecular replacement.

Dear Sudipta,
you are correct, your original scala.mtz has the wrong space group in it, 
resulting in very high Rfactors (and presumably bad electron density).
In these cases, I usually reprocess (remerge) the data in the correct space 
group to get the statistics right (and gain probably a few extra h00 
reflections that got rejected in P212121). If you use the same a, b and c axes 
as before, you do not need to rerun Phaser, otherwise you have to.
If you have translational NCS, you have to live with it. The only way to get 
rid of it is to find another crystal with a different crystal packing.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Sudipta 
Bhattacharyya
Gesendet: Dienstag, 15. Juli 2014 20:32
An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Translational NCS and molecular replacement.

Dear Community,

I have some doubts to clarify. In a way to solve a structure by Phaser-MR, I 
found phaser ended up with a potentially good MR solution (with good 
statistics, packing and electron density, and as we know the homolog structure 
so in a reasonable biological assembly also). However, the space group where 
phaser found the potential solution (P22121) is different what we got in 
pointless (P212121), and phaser also indicated the presence of translational 
NCS (NCS translation vector = 0.500, 0.494 0.391). Now when we try to refine 
the structure with its original scala.mtz file (which is indexed and sclaled in 
P212121) and phaser generated pdb file, the R/Rfree is very high (around 0.5) 
but when I tried refining with mtz file generated by phaser, it was reasonable, 
at least for first cycle of refinement (R/Rfree, 0.41/0.46). Now my question 
is, can I use the phaser generated mtz file instead of the original scala.mtz 
for further refinement? Or I have to reindex my original data into phaser 
suggested spacegroup and run the MR again? Translational NCS are generally 
associated with high R values, is there any way to get rid of that problem?

Best regards,
Sudipta.

[ccp4bb] Translational NCS and molecular replacement.

[Sudipta Bhattacharyya]

Dear Community,

I have some doubts to clarify. In a way to solve a structure by Phaser-MR,
I found phaser ended up with a potentially good MR solution (with good
statistics, packing and electron density, and as we know the homolog
structure so in a reasonable biological assembly also). However, the space
group where phaser found the potential solution (P22121) is different what
we got in pointless (P212121), and phaser also indicated the presence of
translational NCS (NCS translation vector = 0.500, 0.494 0.391). Now when
we try to refine the structure with its original scala.mtz file (which is
indexed and sclaled in P212121) and phaser generated pdb file, the R/Rfree
is very high (around 0.5) but when I tried refining with mtz file generated
by phaser, it was reasonable, at least for first cycle of refinement
(R/Rfree, 0.41/0.46). Now my question is, can I use the phaser generated
mtz file instead of the original scala.mtz for further refinement? Or I
have to reindex my original data into phaser suggested spacegroup and run
the MR again? Translational NCS are generally associated with high R
values, is there any way to get rid of that problem?

Best regards,
Sudipta.

Re: [ccp4bb] translational NCS

[Eleanor Dodson]

pdbset xyzin mol1.pdb xyzout mol1-tran1.pdb
SHIFT frac x,y,z  (where x,y,z is the patterson peak)
end

OR
pdbset xyzin mol1.pdb xyzout mol1-tran2.pdb
SHIFT frac -x,-y,-z  (since -x,-y,-z is also a the patterson peak)
end

Nicolas Soler wrote:

Dear CCP4bbs,

I am dealing with a case involving pseudo-translational symmetry.
I wanted to know what was the simplest way to draw NCS copies of a 
molecule deduced from the positions I observed in native Patterson. Is 
there a translate option where on can give fractional coordinates in 
Coot or Pymol?


Thanks for your help!

Nicolas

[ccp4bb] translational NCS

[Nicolas Soler]

Dear CCP4bbs,

I am dealing with a case involving pseudo-translational symmetry.
I wanted to know what was the simplest way to draw NCS copies of a 
molecule deduced from the positions I observed in native Patterson. Is 
there a translate option where on can give fractional coordinates in 
Coot or Pymol?


Thanks for your help!

Nicolas