[ccp4bb] what is NCS operators of parrot

[Wang]

Hi everyone:
   I used parrot to do density modification, and it found ncs operators. The 
operators found are:

Non-crystallographic operators:

 -ncs-operator 
-144.47,179.729,-144.321,18.7789,16.4179,19.809,16.4383,16.4507,19.809

 -ncs-operator 
-10.0955,179.846,169.903,27.2216,37.7585,38.88,27.0706,40.5231,38.88

  What does the operators mean? Is that Eular angle? But I changed it to 
ratation matrix, the result is not so good.

  Thanks

   Best wishes

 Wang

Re: [ccp4bb] Twinning

[Eleanor Dodson]

Yes - that is true.

Any crystal might be split, and give diffraction with overlapping 
lattices- ie show non-merohedral twinning. If you are lucky/careful you 
might only get a few spots which overlap after integration of one of the 
lattices- not enough to be detected as "twinning" from the statistical 
analysis...


but the SG R3 is a classic one for merohedral twinning with operator k,h,-l

Have you looked at the scala log file - you need to check the L test for 
twinning, whether there is non-crystallographic translation etc.. as 
well as looking at the moment plots.


Eleanor

On 04/06/2011 05:08 PM, ka...@ssl.serc.iisc.ernet.in wrote:

Dear users,

  Is there a possibility that both non-merohedral twinning
and partial hemihedral twinning occur in the same crystal?
In one of the data in R3, sfcheck indicated twinning and thus
upon using twinning option during refinement, refmac considered
twinning operator of (k h -l). But the images also show some
interpenetrating lattice spots which are characteristics of
non-merohedral twinning.

  Probably one of the lattices have been more prominent
and hence detected and processed by mosflm with an overall
Rmerge of 8.2 at 2.35 Ang resolution.

  So is there such a possibility?

Thanking you
With Regards
Kavya

[ccp4bb] XXII IUCr Congress and General Assembly - Madrid (Spain)

[Martin M. Ripoll]

Dear colleagues,

 

This is just to remind you that the “XXII Congress and General Assembly” of
the IUCr (International Union of Crystallography) will be held in Madrid
(Spain) from 22-30 August 2011 and that your Spanish colleagues kindly
invite you to participate not only in the most important crystallographic
event until 2014, but also to enjoy a fruitful meeting in a sunny and full
of life city!

 

All information is to be found in: 

http://www.iucr2011madrid.es/ 

although two important dates to remember are:

April 15, 2011: Deadline for abstracts submission

May 15,  2011: Deadline for "Early bird" registration

 

All the best and see you in Madrid!

 

On behalf of the Organizing Committee,

 

Martin (Vice-Chairman)



Dr. Martin Martinez-Ripoll

Research Professor

  xmar...@iqfr.csic.es

Department of Crystallography & Structural Biology

  www.xtal.iqfr.csic.es

Telf.: +34 917459550

Consejo Superior de Investigaciones Científicas

Spanish National Research Council

www.csic.es

  Sin título-1

 

<>

Re: [ccp4bb] Twinning

[kavya]

> what does the twinning analysis by Phenix tell?
> if you haven't done it yet i would think you should run the processed
> data through phenix Xtriage and see
> what is the twinning analysis tell you.
> It also is a good program to solve your space group issues.
> I think you might need a mtz file or sca as the input.

xtriage had indicated a presence of merohedral twin, with twin law =
h,-h-k,-l

> first of all if you can collect a better data set.
> is non-merohedral and partial hemihedral twinning possibility?
> highly unlikely. chances are you have many small plates of crystals
> growing
> close by and you are collecting data on a bunch of crystals stacked
> up.just
> guessing.
>

It was a single crystal. atleast morphologically it was not looking
like multiple crystal.

> I remember, at an ACA meeting there was a talk discussing twinning issues.
> The gist was: he did process a data set with so complicated twinning
> issues and
> it took several months to get a solution.
> later they were successful in getting better crystals
> and solved the same protein structure in a different space group
> and the electron density map obtained from
> both looked so drastically different the final interpretation of the
> active site function
> was so different.
> question number 1: if the data of such complication is solved how much
> information
> is obtained?
> question number 2: is it possible to get better data than this?
> question number 3: if there is no other way to improve your crystals i
> will
> keep on working on such issues.
>

Fortunately I have got another data, I was able to get a solution for the
twinned structure, but was difficult to refine, When I compared twinned
structure with those two structures it does not vary much.

> Hope this helps
> cheers
> Padayatti PS
>

With Regards

Kavya


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[ccp4bb] shape complementarity using NMR structures

[Sollepura Yogesha]

Hello Everyone,

I am trying to calculate shape complementarity of NMR structure (1U89) using 
CCP4 Sc.

I get following error

 $WARNING: NO CRYST CARDS READ$

 
 $TEXT:Warning: $$ comment $$ 
 WARNING:  NO CRYST CARDS READ FROM XYZIN
 $$


 
 $TEXT:Warning: $$ comment $$ 
 WARNING:  NO SCALE CARDS READ FROM XYZIN
 $$


Any advice is highly appreciated.

Thanks in advance.

Regards,
Yogesha

Re: [ccp4bb] shape complementarity using NMR structures

[Sollepura Yogesha]

Thanks Boaz.
I tried that too. But error was same.
What worked is removal of all models except 1 model and removal of all waters.
Thanks
Yogesha

From: Boaz Shaanan [bshaa...@exchange.bgu.ac.il]
Sent: Thursday, April 07, 2011 5:44 PM
To: Sollepura Yogesha
Subject: RE: shape complementarity  using NMR structures

Hi,

I guess you have to provide a dummy unit cell and identity matrix (which NMR 
structures obviously don't have) in order to make SC happy.

Boaz

Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710




From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sollepura 
Yogesha [yoge...@scripps.edu]
Sent: Friday, April 08, 2011 12:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] shape complementarity  using NMR structures

Hello Everyone,

I am trying to calculate shape complementarity of NMR structure (1U89) using 
CCP4 Sc.

I get following error

 $WARNING: NO CRYST CARDS READ$


 $TEXT:Warning: $$ comment $$
 WARNING:  NO CRYST CARDS READ FROM XYZIN
 $$



 $TEXT:Warning: $$ comment $$
 WARNING:  NO SCALE CARDS READ FROM XYZIN
 $$


Any advice is highly appreciated.

Thanks in advance.

Regards,
Yogesha

[ccp4bb] anisotropy vs TLS

[Kenneth A. Satyshur]

peoples:

I know that TLS is a group B factor for regions of proteins that are moving the 
same.
It is used in low res structures. But at what resolution does one begin 
anisotropic, i.e
individual aniso for each atom, and leave TLS out. Or can one still use TLS to 
first
compensate for large motions and then dampen down the individual atoms with 
aniso ADP?
If both the aniso and TLS are used, how does a person interpret the results? 
What programs
are there to see just what is large body motions and what is atoms.
thanks

--
Kenneth A. Satyshur, M.S.,Ph.D.
Associate Scientist
University of Wisconsin
Madison, Wisconsin 53706
608-215-5207
<>

[ccp4bb] Tev Cleavage issue !!

[anita p]

Hi Crystallographers,
 I am working of 23 Kda protein with a Nterminal  His tag and a TEV cleavage
site.
 I am getting crystals with the his tag and tev site intact, but they dont
diffract.
 *Is it probable that they dont diffract because of the extra his tag and
the tev site?*

 I am trying to get rid of this tag but the reaction is optimum at 10:1
protein to TEV ratio in micrograms overnight incubation without shaking.
 I tried to run it on histrap column after this reaction but I am not able
to purify  cleaved protein from TEV and uncleaved.
 I have tried several times but I get 3 bands ie., the TEV, uncleaved and
Cleaved.
 I have also tried to use the Nibeads instead of the histrap column, but no
difference is seen.
* Is there a possible way to approach this problem?*

 Suggestions awaited
 Anita

Re: [ccp4bb] Tev Cleavage issue !!

[Artem Evdokimov]

For starters, you could re-clone the protein with e.g. just a His tag or
move the tag to another end, or put some distance between the end of TEV
site and the protein; or perhaps use no tag at all -- or a different one?

Is it possible that the tag is messing you up - yes. Is it 'probable' - I
can't say that I know because I've crystallized literally dozens of proteins
with His-tags attached, and more than a few with His-tag and cleavage site.
I prefer a plain His-tag or a cleaved one, to be sure - but I am not quick
to blame the tag (since there are so many other possible things to blame).

Based on the behavior of your protein after cleavage, it may be that you
have oligomer(s) forming in solution such that cleaved and uncleaved
proteins do not segregate. You may wish to explore other kinds of
chromatographic separation e.g. ion exchange of HIC - they may or may not
work out. You can also consider cleaving your protein at lower
concentration, in the presence of detergents or polyols, etc.

Cheers,

Artem

On Thu, Apr 7, 2011 at 9:37 PM, anita p  wrote:

> Hi Crystallographers,
>  I am working of 23 Kda protein with a Nterminal  His tag and a TEV
> cleavage site.
>  I am getting crystals with the his tag and tev site intact, but they dont
> diffract.
>  *Is it probable that they dont diffract because of the extra his tag and
> the tev site?*
>
>  I am trying to get rid of this tag but the reaction is optimum at 10:1
> protein to TEV ratio in micrograms overnight incubation without shaking.
>  I tried to run it on histrap column after this reaction but I am not able
> to purify  cleaved protein from TEV and uncleaved.
>  I have tried several times but I get 3 bands ie., the TEV, uncleaved and
> Cleaved.
>  I have also tried to use the Nibeads instead of the histrap column, but no
> difference is seen.
> * Is there a possible way to approach this problem?*
>
>  Suggestions awaited
>  Anita
>

Re: [ccp4bb] anisotropy vs TLS

[Pavel Afonine]

Hi Kenneth,

I hope this will answer most of your questions:

http://www.phenix-online.org/newsletter/

see "TLS for dummies" and "On atomic displacement parameters..." articles.

Pavel.


On Thu, Apr 7, 2011 at 5:39 PM, Kenneth A. Satyshur wrote:

> peoples:
>
> I know that TLS is a group B factor for regions of proteins that are moving
> the same.
> It is used in low res structures. But at what resolution does one begin
> anisotropic, i.e
> individual aniso for each atom, and leave TLS out. Or can one still use TLS
> to first
> compensate for large motions and then dampen down the individual atoms with
> aniso ADP?
> If both the aniso and TLS are used, how does a person interpret the
> results? What programs
> are there to see just what is large body motions and what is atoms.
> thanks
>
> --
> Kenneth A. Satyshur, M.S.,Ph.D.
> Associate Scientist
> University of Wisconsin
> Madison, Wisconsin 53706
> 608-215-5207
>

Re: [ccp4bb] Twinning

[kavya]

Respected Madam,

> Yes - that is true.
>
> Any crystal might be split, and give diffraction with overlapping
>lattices- ie show non-merohedral twinning. If you are lucky/careful you
>might only get a few spots which overlap after integration of one of
the >lattices- not enough to be detected as "twinning" from the
statistical >analysis...
>
> but the SG R3 is a classic one for merohedral twinning with operator k,h,-l
>
> Have you looked at the scala log file - you need to check the L test for
>twinning, whether there is non-crystallographic translation etc.. as
well >as looking at the moment plots.

I have checked for twinning in L test and H-test both of which indicated
slight twinning. I am attached H-test and L-test graph herewith.

Sfcheck also indicated 21.2% of pseudotranslation along 0.667 0.333 0.000.
But MR using phaser gave a dimer in asu which are related by a 2 fold,
so there was no indication of any pseudo translation and protein
is bound to 10-12 cadmiums. Could this off origin peak be due to the
presence of the heavy atoms? how can i rule out the presence of this
pseudotranslation as indicated in sfcheck?


Thanking you
Respectfully
Kavya

> On 04/06/2011 05:08 PM, ka...@ssl.serc.iisc.ernet.in wrote:
>> Dear users,
>>
>>   Is there a possibility that both non-merohedral twinning
>> and partial hemihedral twinning occur in the same crystal?
>> In one of the data in R3, sfcheck indicated twinning and thus
>> upon using twinning option during refinement, refmac considered
twinning operator of (k h -l). But the images also show some
>> interpenetrating lattice spots which are characteristics of
>> non-merohedral twinning.
>>
>>   Probably one of the lattices have been more prominent
>> and hence detected and processed by mosflm with an overall
>> Rmerge of 8.2 at 2.35 Ang resolution.
>>
>>   So is there such a possibility?
>>
>> Thanking you
>> With Regards
>> Kavya
>>
>>
>
> --
> This message has been scanned for viruses and
> dangerous content by MailScanner, and is
> believed to be clean.
>
>


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<><>

Re: [ccp4bb] anisotropy vs TLS

[Robbie Joosten]

Dear Kenneth,

IMO there is no resolution cut-off to decide to go from TLS to individual 
anisotropic Bs. I use the number of reflections per atom. You are refining 9 
parameters per atom so you need quite a lot. When I have>18 ref/atom I switch 
to anisotropic. I try both isotropic and anisotropic Bs with> 13.5 reflections 
per atom. You need good evidence that the anisotropic model  is better than an 
isotropic model, looking at R-free is not good enough. When you add so many 
parameters R-free will drop anyway. Ethan Merritt discussed a good test for 
this at the CCP4 study weekend. If you use Refmac, I have a tool that uses that 
method to compare the logfiles from too models and helps decide which model is 
best.
Combining TLS and anisotropic Bs is a bit over the top. You could use 
anisotropic Bs and then use TLSMD to extract the bulk movement.

Cheers,
Robbie  

> Date: Thu, 7 Apr 2011 19:39:39 -0500
> From: satys...@wisc.edu
> Subject: [ccp4bb] anisotropy vs TLS
> To: CCP4BB@JISCMAIL.AC.UK
> 
> peoples:
> 
> I know that TLS is a group B factor for regions of proteins that are moving 
> the same.
> It is used in low res structures. But at what resolution does one begin 
> anisotropic, i.e
> individual aniso for each atom, and leave TLS out. Or can one still use TLS 
> to first
> compensate for large motions and then dampen down the individual atoms with 
> aniso ADP?
> If both the aniso and TLS are used, how does a person interpret the results? 
> What programs
> are there to see just what is large body motions and what is atoms.
> thanks
> 
> --
> Kenneth A. Satyshur, M.S.,Ph.D.
> Associate Scientist
> University of Wisconsin
> Madison, Wisconsin 53706
> 608-215-5207
  

Re: [ccp4bb] Tev Cleavage issue !!

[Chen Guttman]

Hey Anita,
I would like to add to Artem's comment that you can also try and cleave the
protein at 30c for 2hr and then continue the cleavage overnight at 4c (you
should check and see that your protein can withstand 30c incubation for 2hr,
of course).
In regard to your non-diffracting crystals - you can try seeding: Streak or
Macro-seed your crystals onto a screen ("screen seed") or onto the same
conditions in which the crystals grew. Sometime you might get different
morphologies that might diffract.
Good luck,
Chen

---
Chen Guttman
The Zarivach laboratory for Macromolecular Crystallography
Building 39, Room 009B
Ben-Gurion University of the Negev
POBox 653
Zip Code 84105
Beer-Sheva
Israel
http://lifeserv.bgu.ac.il/wb/zarivach
Tel. +972-8-6479519
Fax. +972-8-6472970



On Fri, Apr 8, 2011 at 04:37, anita p  wrote:

> Hi Crystallographers,
>  I am working of 23 Kda protein with a Nterminal  His tag and a TEV
> cleavage site.
>  I am getting crystals with the his tag and tev site intact, but they dont
> diffract.
>  *Is it probable that they dont diffract because of the extra his tag and
> the tev site?*
>
>  I am trying to get rid of this tag but the reaction is optimum at 10:1
> protein to TEV ratio in micrograms overnight incubation without shaking.
>  I tried to run it on histrap column after this reaction but I am not able
> to purify  cleaved protein from TEV and uncleaved.
>  I have tried several times but I get 3 bands ie., the TEV, uncleaved and
> Cleaved.
>  I have also tried to use the Nibeads instead of the histrap column, but no
> difference is seen.
> * Is there a possible way to approach this problem?*
>
>  Suggestions awaited
>  Anita
>

[ccp4bb] Regarding Real Space R value

[prakash shukla]

Dear Friends,
I want to calculate RSR value of my protein and Ligand .
   For this I have tried 2fofc.inp through CNS and MAPMAN RS fit
calculation but could not get satisfactory result.
   I will be grateful if any body help me to solve this problem.I will
apppreciate your comment.
   Thanks in advance!
Prakash

Re: [ccp4bb] Twinning

[kavya]

> this can screw up your statistics on twinning and this can be due to
> the NCS rotation
> axis (or will be based on what you say) parallel to a crystallographic
> symmetry axis.
> just check that. it should still refine though but this will have
> opposite effect to twinning,
> so it can be problematic. although its not clear to me what your
> problem is stucture solution
> actually was.
>
> just as a general comment,
>
> tommi

NCS axis is not parallel to any crystallographic axis. Although initially
had some problem in refinement because of twinning. I did not have any
problem in obtaining the solution. So my doubt was that even though there
was an indication of pseudotranslation, why was there no problem in
obtaining the solution? So I wanted to know whether this pseudotranslation
peak was due to the presence of heavy atoms in the structure.


>
>
> Tommi Kajander, Ph.D., Docent
> Macromolecular X-ray Crystallography
> Research Program in Structural Biology and Biophysics
> Institute of Biotechnology
> P.O. Box 65 (Street: Viikinkaari 1, 4th floor)
> University of Helsinki
> FIN-00014 Helsinki, Finland
> Tel. +358-9-191 58903
> Fax  +358-9-191 59940
>
>
> --
> This message has been scanned for viruses and
> dangerous content by MailScanner, and is
> believed to be clean.
>
>



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Re: [ccp4bb] Tev Cleavage issue !!

[Peter Czabotar]

Hi Anita,

We have had success setting up drops with TEV present. We simply added TEV at a 
50:1 molar ratio and then set up the drops a couple of hours later. We went 
from having twinned crystals at 3A to untwinned at 2A, the crystal form also 
changed from orthorhombic to monoclinic, all in the same drop condition. We 
might have just got lucky, but it is an easy experiment to try. 

Cheers
Peter

___
Dr Peter Czabotar
Structural Biology Division
The Walter and Eliza Hall Institute of Medical Research
1G Royal Parade, Parkville, Victoria, Australia
Ph:  (+61 3) 9345 2689
___


On Apr 8, 2011, at 12:37 PM, anita p wrote:

> Hi Crystallographers,
>  I am working of 23 Kda protein with a Nterminal  His tag and a TEV cleavage 
> site.
>  I am getting crystals with the his tag and tev site intact, but they dont 
> diffract.
>  Is it probable that they dont diffract because of the extra his tag and the 
> tev site?
> 
>  I am trying to get rid of this tag but the reaction is optimum at 10:1 
> protein to TEV ratio in micrograms overnight incubation without shaking.
>  I tried to run it on histrap column after this reaction but I am not able to 
> purify  cleaved protein from TEV and uncleaved.
>  I have tried several times but I get 3 bands ie., the TEV, uncleaved and 
> Cleaved.
>  I have also tried to use the Nibeads instead of the histrap column, but no 
> difference is seen.
>  Is there a possible way to approach this problem?
> 
>  Suggestions awaited
>  Anita


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