Re: [ccp4bb] Domain Motion Analysis

[00000c2488af9525-dmarc-request]

Hello Tony, I think CueMol gives you the screw rotation and translation when you fit domains. Jon CooperOn 8 Jul 2020 18:53, Eleanor Dodson <176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:Well - I use standard software.Use GESANT or some such program to fit Structure 2 A to Structure ! A only - output wilh have all A B C but only A has been fitted//Now fit Structure 2 B from fitted structure to Structur 1 B .It will give you a PHI CHI Kappa needed to rotate B2 to B1 Is that clear enough - I can send an example..EleanorOn Wed, 8 Jul 2020 at 18:09, kazi nasrin sultana  wrote:Hi,You can try elNemo server.Regards,NasrinPhD scholarOn Wed, Jul 8, 2020, 10:16 PM Antony Oliver  wrote:







Dear CCP4bb
 
Is anyone kindly able to point me at a server or recommend software that might help me analyse relative domain movements – by comparison of two structures?
 
Structure 1 has molecules A and B. 

Structure 2 has molecules A, B and C.
 
I am interested in the induced movements, and a description thereof, that the presence of molecule C produces in molecules A and B.
 
I am aware of DynDom – but I can’t seem to make it produce a readily interpretable output (undoubtedly my fault).
 
Many thanks for your help in advance.
 
Antony.
 


 

Antony W Oliver
FHEA, PhD
Faculty Senior Research Fellow
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

 





(office): +44 (0)1273 678349
(lab): +44 (0)1273 677512

 

antony.oliver@sussex.ac.uk
https://www.sussex.ac.uk/lifesci/oliverlab
https://tinyurl.com/aw-oliver
https://orcid.org/-0002-2912-8273

http://www.sussex.ac.uk/lifesci/internal/staff/support



 
 
 


 





To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

Re: [ccp4bb] Protein expression (codon bias)

[00000c2488af9525-dmarc-request]

I did crystallise a protein expressed in M. smegmatis a while ago (early 90's)! The cloning was done by Ying Zhang:https://www.jhsph.edu/faculty/directory/profile/786/ying-zhangIt might be worth dropping him a line. That's all I can suggest, sorry!!Good luck.Jon CooperOn 7 Jul 2020 10:07, Matthew Snee  wrote:

Hi all




I'm designing genes for _expression_ in M. smegmatis (safe host for Mtb proteins), but its not possible (or advisable due to the GC content) to optimise for

mycobacterial _expression_.




Would anyone with experience be able to tell me if its fine to stick with the E. coli codon optimisation, or if there is an advantage going with b. subtilis (or another G+ organism that is in Thermo's list).




Best wishes




Matthew.



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

Re: [ccp4bb] number of frames to get a full dataset?

[00000c2488af9525-dmarc-request]

I think it is quite an interesting question in principle for Laue crystallography (now probably only relevant in the neutron world?) since, for example, if one had a crystal in the 432 point group, you could collect an essentially complete dataset with one 'image'. Given that each image can take several hours to collect, the number of them would seem to be important...Jon CooperOn 30 Jun 2020 20:24, Gerard Bricogne  wrote:Dear Ed,

 Concerning your remark that "use of terms redundancy and multiplicity
to describe the same concept is by itself redundant", one could perhaps say
that redundancy is an abstract property of a dataset, while multiplicity is
a numerical attribute. Redundancy is desirable because if some measurements
turn out to be corrupted, there are spare ones to salvage completeness. In
this form it is an aspect of quality, without being in itself a numerical
entity. That property of redundancy is conferred by high multiplicity of
measurement, which is very much a numerical entity. The two terms are
threfore not redundant, but are made so in practice by the shorthand of
giving the numerical attribute the name of the abstract property it gives
rise to.

 Regarding the relation to replication, I can remember Peter Mueller's
book on refinement with SHELX quoting George Sheldrick's point that simply
repeating a measurement is of limited usefulness, because one repeats its
systematic errors, and advocating that what is truly valuable is to make
multiple measurements in conditions such that their systematic errors are
likely to be different. This diversity of conditions gives rise to what he
called "true multiplicity". There is clearly a close affinity between this
concept and that of redundancy viewed as a protection against corrupted
individual measurements.

 The essential thing, though, is not the choice of terminology but the
practical decision of abjuring the pernicious mindset alluded to by the
Subject line of this thread :-) .


 With best wishes,

  Gerard.

--
On Tue, Jun 30, 2020 at 11:19:38AM -0400, Edwin Pozharski wrote:
> Replicate is a good option with its own problems as it can be seen as
> referring to exact copy which multiple measurements clearly aren't. It does
> have an advantage of being the word used by non-crystallographers though.
> 
> As a lame attempt at joke, use of terms redundancy and multiplicity to
> describe the same concept is by itself redundant.
> 
> On Mon, Jun 29, 2020, 7:21 PM Bernhard Rupp 
> wrote:
> 
> > Ah…the rise of the replicants …
> >
> >
> >
> > https://www.youtube.com/watch?v=yWPyRSURYFQ
> >
> >
> >
> > …and don’t forget the and the Voight-Kampff Test results in Table 1.
> >
> >
> >
> > Best, BR
> >
> >
> >
> > *From:* Pierre Rizkallah 
> > *Sent:* Monday, June 29, 2020 15:46
> > *To:* b...@hofkristallamt.org; CCP4BB@JISCMAIL.AC.UK
> > *Subject:* RE: [ccp4bb] number of frames to get a full dataset?
> >
> >
> >
> > You’re missing out on a grand opportunity for iconoclasm here. Try out
> > ‘Degree of Replication’ or ‘Average Replication’ or ‘Replicate Frequency’.
> > Any other offerings!
> >
> >
> >
> > P.
> >
> > ***
> >
> > Dr Pierre Rizkallah, Senior Lecturer Structural Biology
> >
> > Institute of Infection & Immunology, Sir Geraint Evans Building,
> >
> > School of Medicine, Heath Campus, Cardiff, CF14 4XN
> >
> > email: rizkall...@cardiff.ac.uk    phone: +44 29 2074 2248
> >
> > http://www.cardiff.ac.uk/people/view/126690-rizkallah-pierre
> >
> >
> >
> > *From:* CCP4 bulletin board  *On Behalf Of *Bernhard
> > Rupp
> > *Sent:* 29 June 2020 23:36
> > *To:* CCP4BB@JISCMAIL.AC.UK
> > *Subject:* Re: [ccp4bb] number of frames to get a full dataset?
> >
> >
> >
> > I think it is time to escalate that discussion to crystallographic
> > definition purists like Massimo or to a logical consistency proponent like
> > Ian who abhors definitional vacuum 😊
> >
> >
> >
> > Cheers, BR
> >
> >
> >
> > *From:* CCP4 bulletin board  *On Behalf Of *Andreas
> > Förster
> > *Sent:* Monday, June 29, 2020 15:24
> > *To:* CCP4BB@JISCMAIL.AC.UK
> > *Subject:* Re: [ccp4bb] number of frames to get a full dataset?
> >
> >
> >
> > I like to think that the reflections I carefully measured at high
> > multiplicity are not redundant, which the dictionary on my computer defines
> > as "not or no longer needed or useful; superfluous" and the American
> > Heritage Dictionary as "exceeding what is necessary or natural;
> > superfluous" and "needlessly repetitive; verbose".
> >
> >
> >
> > Please don't use the term Needless repetitivity in your Table 1.  It sends
> > the wrong message.  Multiplicity is good.
> >
> >
> >
> > All best.
> >
> >
> >
> >
> >
> > Andreas
> >
> >
> >
> >
> >
> >
> >
> > On Tue, Jun 30, 2020 at 12:03 AM James Holton  wrote:
> >
> > I have f

Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?

[00000c2488af9525-dmarc-request]

Hello JohnDoes the IUCr dictionary list 'degeneracy'?Jon CooperOn 30 Jun 2020 17:11, Gerard Bricogne  wrote:Dear Bernhard,



 That is true, and the discrepancies between repeated measurements of

the same hkl would have to be parametrised differently from those between

symmetry-related ones (e.g. in terms of radiation damage only, while the

others would also involve absorption effects). However I am not aware that

the existing data processing programs we use actually make and exploit this

distinction.



 Going back to the initial topic of this thread, the main take-home

lesson for Murpholino should be: preoccupations about minimising the number

of frames to get completeness belong to a now obsolete age - instead use the

new paragigm of high-(redundancy/multiplicity) data collection with a low

transmission so that you can spread the dose your crystal can withstand over

the requisite angular range. No matter how you call the "abundance" property

of your final dataset, make sure it is high!



 The case of low symmetry has been mentioned: the extra guidance for

Murpolino is that if you are in P1, you will never get completeness with a

single orientation, so make sure that you use a multi-axis goniometer and

collect data in at least two sufficiently different orientations.





 With best wishes,



  Gerard.



--

On Tue, Jun 30, 2020 at 08:49:53AM -0700, Bernhard Rupp wrote:

> .…but there is a difference whether I measure the same identical hkl over again or ‘preferably in more than one symmetry-equivalent position’, to quote the

> 

> IUCr. So do we have a MPSR for the same reflection and a MPRR for the related reflections?

> 

>  

> 

> Cacophonically yours,

> 

>  

> 

> BR

> 

>  

> 

> From: CCP4 bulletin board  On Behalf Of John R Helliwell

> Sent: Tuesday, June 30, 2020 08:36

> To: CCP4BB@JISCMAIL.AC.UK

> Subject: Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?

> 

>  

> 

> Dear Herman,

> 

> I think that MPR is a very neat and tidy, excellent, proposal.

> 

> Moreover it uses the word “measurements”, and we are an experimental based science.

> 

> I support it.

> 

> Great.

> 

> Greetings,

> 

> John 

> 

> Emeritus Professor John R Helliwell DSc

> 

>  

> 

>  

> 

>  

> 

>  

> 

> On 30 Jun 2020, at 15:10, Schreuder, Herman /DE  > wrote:

> 

>  

> 

> Dear BB,

> 

>  

> 

> Since there does not seem a generally accepted term for the subject of this discussions, and since even the IUCR scriptures do not give any guidance, I would propose to introduce a completely new term:

> 

>  

> 

> Measurements per reflection or MPR

> 

>  

> 

> This term is politically neutral, should adequately describe this particular statistic and is not associated with entrenched traditions at either side of the Atlantic.

> 

>  

> 

> What do you think?

> 

> Herman

> 

>  

> 

>  

> 

> Von: CCP4 bulletin board  > Im Auftrag von John R Helliwell

> Gesendet: Dienstag, 30. Juni 2020 14:34

> An: CCP4BB@JISCMAIL.AC.UK  

> Betreff: [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?

> 

>  

> 

> EXTERNAL : Real sender is owner-ccp...@jiscmail.ac.uk   

> 

>  

> 

> Dear Colleagues,

> 

> In an effort to break this naming deadlock, and with Massimo and Ian not showing up as yet, I checked the IUCr Dictionary.

> 

> “Redundancy“ and “Multiplicity“ are not listed.

> 

> The more generic term “Statistical Descriptors“ is though and even offers Recommendations:-

> 

> http://ww1.iucr.org/iucr-top/comm/cnom/statdes/recomm.html  

> 

> Point 1, first sentence, fits the various wishes of this thread succinctly, if not in a single word, and even not readily allowing an easy acronym. 

> 

> Greetings,

> 

> John 

> 

>  

> 

> Emeritus Professor John R Helliwell DSc

> 

>  

> 

>  

> 

> 

> 

> 

> 

> On 30 Jun 2020, at 13:11, Phil Jeffrey  > wrote:

> 

> The people that already use multiplicity are going to find reasons why it's the superior naming scheme - although the underlying reason has a lot to do with negative associations with 'redundant', perhaps hightened in the current environment.  And conversely redundant works for many others - Graeme's pragmatic defense of multiplicity actually works both ways - any person who takes the trouble to read the stats table, now exiled to Supplementary Data, knows what it means.  Surely, then, the only way forward on this almost totally irrelevant discussion is to come up with a universally-loathed nomenclature that pleases nobody, preferably an acronym whose origins will be lost to history and the dusty CCP4 archives (which contain threads similar to this one).  I humbly submit:

> 

> NFDOF: Nearly Futile Data Overcollection Factor ?

> [*]

> 

> Or, even better, could we not move on to equally pointless discussions of the inappropriateness of "R-factor" ?  I have a long history of rearguard action trying to give stupid acronyms a wider audien

Re: [ccp4bb] I cannot download xds

[00000c2488af9525-dmarc-request]

With wget you can pretend to be somewhere else with the: --referer "http://www../"option. You could even put in the heidelberg url and then the server thinks your request comes from there ;-0Might help ;-?Jon CooperOn 29 Jun 2020 02:17, Murpholino Peligro  wrote:Dear Kay, I can download anything from ftp://ftp.wwpdb.org/pub/pdb/data/structures/divided/mmCIF/ and it is ftp. Something else I guess.ThanksEl dom., 28 de jun. de 2020 a la(s) 14:43, Kay Diederichs (kay.diederi...@uni-konstanz.de) escribió:The problem could be that your current network / firewall does not allow FTP transfer, whereas it does allow HTTP.
HTH
Kay


On Sun, 28 Jun 2020 13:56:34 -0500, Murpholino Peligro  wrote:

>Dear Gianluca and bogbasic, the provided link will send  to a page where if
>you click on "XDS-INTEL64_Linux_x86_64.tar.gz
>"
>it will redirect you to
>ftp://ftp.mpimf-heidelberg.mpg.de/pub/kabsch/XDS-INTEL64_Linux_x86_64.tar.gz,
>which was what I already did (from the command line with wget, as suggested
>by Folmer, or aria2c, which are two command line programs to download stuff
>from the web)...
>
>Dear Folmer, I might be a bit unlucky.
>I can download other things (PDFs, some raw frames, etc). Is not my Fedora
>as I get the same result from my girlfriend's macbook and my cell phone
>(png attached).
>
>Dear all, I still can not download XDS from
>ftp://ftp.mpimf-heidelberg.mpg.de/pub/kabsch/XDS-INTEL64_Linux_x86_64.tar.gz,
>but if I try from the wiki page
>https://strucbio.biologie.uni-konstanz.de/pub/xds/XDS-INTEL64_Linux_x86_64.tar.gz
>it downloads the compressed folder without any problems.
>
>El dom., 28 de jun. de 2020 a la(s) 10:49, 
>escribió:
>
>> A quick Google search for "download xds" will lead you to this page
>>
>> http://xds.mpimf-heidelberg.mpg.de/html_doc/downloading.html
>>
>>
>>
>> On June 28, 2020 2:39:56 AM GMT+02:00, Murpholino Peligro <
>> murpholi...@gmail.com> wrote:
>>>
>>> ```
>>> ~/Downloads$ aria2c
>>> ftp://ftp.mpimf-heidelberg.mpg.de/pub/kabsch/XDS-INTEL64_Linux_x86_64.tar.gz
>>>
>>> 06/27 19:36:10 [NOTICE] Downloading 1 item(s)
>>> [#3065b2 0B/10MiB(0%) CN:1 DL:0B]
>>> ```
>>>
>>> It stays there forever.
>>> Is there an alternative download url?
>>>
>>>
>>> Thanks
>>>
>>> --
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>>>
>>
>> --
>> Sent from my Android device with K-9 Mail. Please excuse my brevity.
>>
>
>
>
>To unsubscribe from the CCP4BB list, click the following link:
>https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>
>This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
>



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

Re: [ccp4bb] I cannot download xds

[00000c2488af9525-dmarc-request]

Hello, it downloaded to my phone from the link on this page:http://xds.mpimf-heidelberg.mpg.de/html_doc/downloading.htmlIt looks alright: http://u.cubeupload.com/jbcooper/20200628034613.jpgJon CooperOn 28 Jun 2020 01:39, Murpholino Peligro  wrote:```~/Downloads$ aria2c ftp://ftp.mpimf-heidelberg.mpg.de/pub/kabsch/XDS-INTEL64_Linux_x86_64.tar.gz06/27 19:36:10 [NOTICE] Downloading 1 item(s)[#3065b2 0B/10MiB(0%) CN:1 DL:0B]    ```It stays there forever.Is there an alternative download url?Thanks


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

Re: [ccp4bb] number of frames to get a full dataset?

[00000c2488af9525-dmarc-request]

Phil Jefferey was right about the point group (432), which looks like it represents about 0.7 % of the PDB! I tested the completeness with MOSFLM strategy for various sporadic missetting angles and 11 degrees of data does give you around 90 % completeness or more with redundancy close to 2, assuming no detector offsets. Jon CooperOn 23 Jun 2020 16:10, bogba...@yahoo.co.uk wrote:Someone told me there is a cubic space group where you can get away with something like 11 degrees of data. It would be interesting if that's correct. These minimum ranges for data collection rely on the crystal being pre-oriented, which is unheard-of these days, although they can help if someone is nagging you to get off the beam line or if your diffraction fades quickly. Going for 180 degrees always makes sense for a well-behaved crystal, or 360 degrees if you want super anomalous differences. Hope this helps a bit. Jon CooperOn 23 Jun 2020 07:29, Andreas Förster  wrote:Hi Murpholino,in my opinion (*), the question is neither number of frames nor degrees.  The only thing that matters to your crystal is dose.  How many photons does your crystal take before it dies?  Consequently, the question to ask is How best to use photons.  Some people have done exactly that.https://doi.org/10.1107/S2059798319003528All best.Andreas(*) Disclaimer:  I benefit when you use PILATUS or EIGER - but I want you to use them to your advantage.On Tue, Jun 23, 2020 at 12:04 AM Murpholino Peligro  wrote:Hi. Quick question... I have seen *somewhere* that to get a 'full dataset we need to collect n frames':at least 180 frames if symmetry is Xat least 90 frames if symmetry is Yat least 45 frames if symmetry is ZCan somebody point where is *somewhere*? ...also...what other factors can change n... besides symmetry and radiation damage?Thanks


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

-- Andreas Förster, Ph.D.Application Scientist Crystallography, Area Sales Manager Asia & Pacific	Phone: +41 56 500 21 00 | Direct: +41 56 500 21 76 | Email: andreas.foerster@dectris.comDECTRIS Ltd. | Taefernweg 1 | 5405 Baden-Daettwil | Switzerland | www.dectris.com    Confidentiality Note: This message is intended only for the use of the named recipient(s)and may contain confidential and/or privileged information. If you are not the intendedrecipient, please contact the sender and delete the message. Any unauthorized use ofthe information contained in this message is prohibited.


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

Re: [ccp4bb] number of frames to get a full dataset?

[00000c2488af9525-dmarc-request]

Well, it can still help. I used to be a great fan of inverse-beam expts! Oh, and some of us prefer the word 'multiplicity' ;-0Jon CooperOn 23 Jun 2020 22:04, "Peat, Tom (Manufacturing, Parkville)"  wrote:

I would just like to point out that for those of us who have worked too many times with P1 or P21 that even 360 degrees will not give you 'super' anomalous differences.



I'm not a minimalist when it comes to data- redundancy is a good thing to have. 


cheers, tom 










Tom Peat
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.peat@csiro.au 












From: CCP4 bulletin board  on behalf of 0c2488af9525-dmarc-request@JISCMAIL.AC.UK <0c2488af9525-dmarc-request@JISCMAIL.AC.UK>
Sent: Wednesday, June 24, 2020 1:10 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] number of frames to get a full dataset?
 


Someone told me there is a cubic space group where you can get away with something like 11 degrees of data. It would be interesting if that's correct. These minimum ranges for data collection rely on the crystal being pre-oriented, which is
 unheard-of these days, although they can help if someone is nagging you to get off the beam line or if your diffraction fades quickly. Going for 180 degrees always makes sense for a well-behaved crystal, or 360 degrees if you want super anomalous differences.
 Hope this helps a bit. 

Jon Cooper


On 23 Jun 2020 07:29, Andreas Förster  wrote:

Hi Murpholino,


in my opinion (*), the question is neither number of frames nor degrees.  The only thing that matters to your crystal is dose.  How many photons does your crystal take before it dies?  Consequently, the question to ask is How best to use photons.  Some
 people have done exactly that.
https://doi.org/10.1107/S2059798319003528

All best.




Andreas




(*) Disclaimer:  I benefit when you use PILATUS or EIGER - but I want you to use them to your advantage.







On Tue, Jun 23, 2020 at 12:04 AM Murpholino Peligro <murpholinox@gmail.com> wrote:


Hi. 
Quick question...
I have seen *somewhere* that to get a 'full dataset we need to collect n frames':
at least 180 frames if symmetry is X

at least 90 frames if symmetry is Y

at least 45 frames if symmetry is Z

Can somebody point where is *somewhere*? 



...also...

what other factors can change n... besides symmetry and radiation damage?



Thanks





To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1





-- 


Andreas Förster, Ph.D.
Application Scientist Crystallography, Area Sales Manager Asia & Pacific

Phone: +41 56 500 21 00 | Direct: +41
 56 500 21 76 | Email: andreas.foerster@dectris.com

DECTRIS Ltd. | Taefernweg
 1 | 5405 Baden-Daettwil | Switzerland | www.dectris.com




    






Confidentiality Note: This message is intended only for the use of the named recipient(s)
and may contain confidential and/or privileged information. If
 you are not the intended
recipient, please contact the sender and delete the
 message. Any unauthorized use of
the information contained in this message
 is prohibited.








To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1







To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1







To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

Re: [ccp4bb] number of frames to get a full dataset?

[00000c2488af9525-dmarc-request]

Someone told me there is a cubic space group where you can get away with something like 11 degrees of data. It would be interesting if that's correct. These minimum ranges for data collection rely on the crystal being pre-oriented, which is unheard-of these days, although they can help if someone is nagging you to get off the beam line or if your diffraction fades quickly. Going for 180 degrees always makes sense for a well-behaved crystal, or 360 degrees if you want super anomalous differences. Hope this helps a bit. Jon CooperOn 23 Jun 2020 07:29, Andreas Förster  wrote:Hi Murpholino,in my opinion (*), the question is neither number of frames nor degrees.  The only thing that matters to your crystal is dose.  How many photons does your crystal take before it dies?  Consequently, the question to ask is How best to use photons.  Some people have done exactly that.https://doi.org/10.1107/S2059798319003528All best.Andreas(*) Disclaimer:  I benefit when you use PILATUS or EIGER - but I want you to use them to your advantage.On Tue, Jun 23, 2020 at 12:04 AM Murpholino Peligro  wrote:Hi. Quick question... I have seen *somewhere* that to get a 'full dataset we need to collect n frames':at least 180 frames if symmetry is Xat least 90 frames if symmetry is Yat least 45 frames if symmetry is ZCan somebody point where is *somewhere*? ...also...what other factors can change n... besides symmetry and radiation damage?Thanks


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

-- Andreas Förster, Ph.D.Application Scientist Crystallography, Area Sales Manager Asia & Pacific	Phone: +41 56 500 21 00 | Direct: +41 56 500 21 76 | Email: andreas.foerster@dectris.comDECTRIS Ltd. | Taefernweg 1 | 5405 Baden-Daettwil | Switzerland | www.dectris.com    Confidentiality Note: This message is intended only for the use of the named recipient(s)and may contain confidential and/or privileged information. If you are not the intendedrecipient, please contact the sender and delete the message. Any unauthorized use ofthe information contained in this message is prohibited.


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

Re: [ccp4bb] AW: Molecular replacement problem

[00000c2488af9525-dmarc-request]

Robert, it would be very interesting to know your cell dimensions and corresponding N. mol. per AU to see if it's trying to be another space group. How long did the crystals take to grow? You could also try Contaminer and Simbad ;-)Jon CooperOn 18 Jun 2020 14:38, "Schreuder, Herman /DE"  wrote:

Dear Robert,
 
In addition to the remarks and suggestions by others: How do your electron density maps look like? Do they look remotely reasonable, or do they look like they had gone through a meat
 grinder? If they look remotely ok, you could also try manual rebuilding, pruning etc. However, if they look chopped, there is no sense in trying to (re)build anything in it, either manually or automatically. I guess you tried all permutations and variations
 of P2221, with zero, one and two screw axes in all positions?
 
Best, 

Herman
 


Von: CCP4 bulletin board  Im Auftrag von
Robert S Phillips
Gesendet: Donnerstag, 18. Juni 2020 15:01
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Molecular replacement problem


 

EXTERNAL : Real sender is
owner-ccp...@jiscmail.ac.uk 

 


I've been pulling out my hair with this for a few months now.  I have data sets to 2.6 A for a new enzyme in the aminotransferase superfamily.  Unfortunately, the
 closest structure is only 25% identity.  MR with PHASER using the monomer was a complete failure.  Since the minimum structure of enzymes in the family is a dimer (the active site is formed at the monomer-monomer interface), I used dimers for MR with PHASER. 
 Most of the results were marginal, but one looks good.  However, it will not refine.  Everything I have done with this solution has failed, simulated annealing, morphing, etc., it will not refine; AUTOBUILD and BUCCANEER with it do not give any useable models,
 since they have poor statistics and low completeness.  The output from PHASER is below.


 


** SINGLE solution

 


** Solution written to PDB file:  DGL_phaser.1.pdb


** Solution written to MTZ file:  DGL_phaser.1.mtz


   Solution annotation (history):


   SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1 PAK=0 LLG=994 TFZ==20.1


   SOLU SPAC P 2 2 21


   SOLU 6DIM ENSE ense_1 EULER  360.0    0.0    0.0 FRAC -0.00 -0.00 -0.00 BFAC -0.12 MULT 2 #TFZ==20.1


   SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89

 


With LLG = 994 and TFZ = 20.1, isn't this a real solution?


 


Robert S. Phillips






Professor of Chemistry and of Biochemistry and Molecular Biology 


University of Georgia

Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphill...@chem.uga.edu
Web:  
http://tryptophan.net






 



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1





To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

Re: [ccp4bb] Question about small molecule crystallography

[00000c2488af9525-dmarc-request]

Re: "it turns out to be very very easy to exceed the count rate where the detector electronics can keep up."Sorry if this is obvious, but I take it you mean "_can't_" keep up?Jon CooperOn 4 Jun 2020 13:06, "Winter, Graeme (DLSLtd,RAL,LSCI)"  wrote:
Dear All,


A small word of caution regarding chemical crystallography on an MX-like beamline - if you have a bright source, a well diffracting crystal and a pixel array detector it is perfectly possible to lose counts in the strongest reflections without
 noticing - certainly without going over the nominal detector count limits if your mosaic spread is very small


At Diamond we faced this issue with i19, which is a dedicated chemical crystallography beamline on an undulator source, with a Pilatus 2 detector - it turns out to be very very easy to exceed the count rate where the detector electronics can keep
 up. This is somewhat less of a problem with Pilatus 3 and Eiger 2X detectors.


So: I would strongly suggest really turning down the intensity on the beam, use fine slicing and be prepared to check that the data are OK before removing your sample. You can solve and refine the structure with e.g. SHELX and get an F^2 calc
 vs. I ops plot which would show systematically under measured low angle reflections. 


We also have a tool in development to bolt on to DIALS called screen19 which estimates the true peak photon rate from a “quick screening run” to offer insights into data collection options - https://github.com/xia2/screen19 -
 this is not perfect but you could find it useful. Our chemical crystallography users certainly find it helpful for planning their experiments. 


Best wishes Graeme


On 4 Jun 2020, at 10:51, Alker, Andre M. <4599f25026c0-dmarc-requ...@jiscmail.ac.uk> wrote:



Dear Jiyuan,


maybe I can add something from the small molecule crystallographer view :-)


Crystallization:
For crystallization of small molecules you normally use solvents and water or mixtures as mentioned already before. At my lab the most successful way to crystallise is evaporation. Please use small glass vials (no plastic, some solvents
 will solute them too). 


You can also use crystallization kits. Here you can use Bernhard Spinglers kit and or the Hampton kit. The Hampton kit didn't work so well for my molecules. I also bought Bernhard's kit but had no time to test it so far. I have to say
 I'm only using crystallization kits at the moment as last try. Maybe that changes in the future.


Measurement: 
Of course you can use the synchrotron for small molecules at the protein beamline. I'm using the PXII protein beamline at the SLS (Swiss light source) for my small molecules if the crystals are too small for my inhouse system or there is a problem
 with my inhouse system. To get the resolution you need for structure solution and a good refinement you have to change the wavelength to 0.7Å. Collect always at least 360 degrees  in steps of 0.5 degrees or smaller. Use high filters and short exposure times
 to secure your crystal against radiation damage. Normally 10 to 20% of the beam and 0.01 to 0.1 sec work fine depending on the crystal size. Do the measurement at low temperatures as usual for protein crystals. Here are my parameters for the SLS-PXII beamline
 equipped with an Eiger detector (wavelength 0.7Å, biggest aperture, 20% transmission, step 0.1 deg/0.01 sec, 1800 degrees, 100K).


Processing, structure solution and refinement:
I process the eiger data with XDS, 
for structure solution I'm using shelxs or shelxd, 
for structure refinement shelxl. 


But as mentioned before there are a lot of programs doing processing, structure solution and refinement. I just started to use Olex as well for solution and refinement. In very earlier times I also used hkl2000 for data processing but
 in my opinion XDS is doing a very good job for small molecule data.


Last but not least I deeply agree with Navdeep to read Mueller et al.'s excellent book about Crystal Structure Refinement. 


Hope this helps a little bit :-)


Best regards,
André



























































André Alker
Senior Scientist, pCMC Analytics
Roche Pharmaceutical Research and Early Development
Roche Innovation Center Basel

F. Hoffmann-La Roche Ltd
Grenzacherstrasse 124
4058 Basel, Switzerland
Phone: +41-61-6880935
email: andre_m.al...@roche.com



Upcoming absences: 
...


Hinweis:
Der Inhalt dieser E-Mail kann vertrauliche Angaben enthalten, die nur für den (die) namentlich genannten Empfänger bestimmt sind. Falls Sie nicht der Adressat dieser E-Mail sind, nehmen Sie Verbindung mit dem Absender auf
 und löschen Sie diese Mitteilung. Jede unbefugte Verwendung der in dieser Mitteilung enthaltenen Informationen ist untersagt.
This message is intended only for the use of the named recipient(s) and may contain confidential and/or privileged information. If you are not the intended recipient, please contact the sender and delete this message. Any unauthorized use of the in

Re: [ccp4bb] Space group/Unit cell

[00000c2488af9525-dmarc-request]

I don't know about LCP but it sounds like a case for Contaminer or Simbad.Jon CooperOn 22 May 2020 11:08, "Demou, Maria"  wrote:

Dear all,

I have a question that may have a straight forward answer, and was wondering if this is a common issue. We have a protein crystallised in I222 space group. This is CRP, but the monomer/pentamer is not predicted to fit in this space group. Is there a possibility
 of the lipid cubic phase being crystallised on it's own, or is there any other obvious reason?




Thank you,

Maria 






To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] How to compare between electron density maps?

[00000c2488af9525-dmarc-request]

Hello, you certainly could normalise maps (i.e. have a map of (density minus average)/rms) with mapman in the Uppsala suite and you can ask Phenix to make the electron electron density normalised when it generates a map (set "map scaling" to "sigma"). The web suggests you can do it with XDLmapman, if you can get hold of it since its deprecated. I wrote a 'jiffy' to do it for ccp4 and x-plor maps but the output is x-plor format. Drop me a line if any interest (or just google for "maidaid map ucl" and the page comes up as top hit). Mapman also used to allow maps to be compared by correlation coefficient. Probably I missed the point of your question but hope some of this helps.Jon CooperOn 11 May 2020 23:18, Murpholino Peligro  wrote: I want to compare electron density features of map A from protein A and map B from protein B...Because each map has a different rmsd level... ...what is the best way to compare electron density between maps?Is there a way to normalize maps or something like that?Thanks


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

On 11 May 2020 23:18, Murpholino Peligro  wrote: I want to compare electron density features of map A from protein A and map B from protein B...Because each map has a different rmsd level... ...what is the best way to compare electron density between maps?Is there a way to normalize maps or something like that?Thanks


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

On 11 May 2020 23:18, Murpholino Peligro  wrote: I want to compare electron density features of map A from protein A and map B from protein B...Because each map has a different rmsd level... ...what is the best way to compare electron density between maps?Is there a way to normalize maps or something like that?Thanks


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

On 11 May 2020 23:18, Murpholino Peligro  wrote: I want to compare electron density features of map A from protein A and map B from protein B...Because each map has a different rmsd level... ...what is the best way to compare electron density between maps?Is there a way to normalize maps or something like that?Thanks


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

On 11 May 2020 23:18, Murpholino Peligro  wrote: I want to compare electron density features of map A from protein A and map B from protein B...Because each map has a different rmsd level... ...what is the best way to compare electron density between maps?Is there a way to normalize maps or something like that?Thanks


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] disinfecting keyboards

[00000c2488af9525-dmarc-request]

Re: injecting disinfect[ing]ant[?] through the USB cable [port?].Dear TimI am intrigued, seriously.Best wishes.Jon CooperOn 29 Apr 2020 19:53, Tim Gruene  wrote:Dear all,



can you make suggestions for how to disinfect computer keyboards, and

instrument panels?



Our facility is going to reboot next week, with shifts so that people

don't meet. The main interface will be the computer keyboards, as well

as the door of our X-ray diffractometer and the mounting of the

crystals.



The keyboard labels may not like alcohols (and the efficiency of

injecting disinfecting through the USB cable is also under discussion,

so I heard).



One way would be to use individual keyboards, and wearing gloves for

replugging, and to use gloves for mounting crystals.



But maybe there are other ways that won't require gloves?



Best regards,

Tim



-- 

--

Tim Gruene

Head of the Centre for X-ray Structure Analysis

Faculty of Chemistry

University of Vienna



Phone: +43-1-4277-70202



GPG Key ID = A46BEE1A







To unsubscribe from the CCP4BB list, click the following link:

https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1


On 29 Apr 2020 19:53, Tim Gruene  wrote:Dear all,



can you make suggestions for how to disinfect computer keyboards, and

instrument panels?



Our facility is going to reboot next week, with shifts so that people

don't meet. The main interface will be the computer keyboards, as well

as the door of our X-ray diffractometer and the mounting of the

crystals.



The keyboard labels may not like alcohols (and the efficiency of

injecting disinfecting through the USB cable is also under discussion,

so I heard).



One way would be to use individual keyboards, and wearing gloves for

replugging, and to use gloves for mounting crystals.



But maybe there are other ways that won't require gloves?



Best regards,

Tim



-- 

--

Tim Gruene

Head of the Centre for X-ray Structure Analysis

Faculty of Chemistry

University of Vienna



Phone: +43-1-4277-70202



GPG Key ID = A46BEE1A







To unsubscribe from the CCP4BB list, click the following link:

https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1


On 29 Apr 2020 19:53, Tim Gruene  wrote:Dear all,

can you make suggestions for how to disinfect computer keyboards, and
instrument panels?

Our facility is going to reboot next week, with shifts so that people
don't meet. The main interface will be the computer keyboards, as well
as the door of our X-ray diffractometer and the mounting of the
crystals.

The keyboard labels may not like alcohols (and the efficiency of
injecting disinfecting through the USB cable is also under discussion,
so I heard).

One way would be to use individual keyboards, and wearing gloves for
replugging, and to use gloves for mounting crystals.

But maybe there are other ways that won't require gloves?

Best regards,
Tim

-- 
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis
Faculty of Chemistry
University of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] neg density/high B on sidechains

[00000c2488af9525-dmarc-request]

Hello, it depends a bit on the resolution. With d<1.2 A you can, of course, do some quite complicated modelling of alternate confirmations, group occupancies, etc - actually you can do this very meaningfully at worse resolutions, too. My favourite subject - if your resolution is better than ~1.4 to 1.6 A, you can often clean up the difference map by refining anisotropic B's. Otherwise (my opinion is that) it's best not to do anything too complicated so just letting the B-factors go a bit high seems the best way! What side chains are we talking about - Cys, Met? I am not an expert on radiation damage but about 20 years ago the discussion was that the more exotic damage e.g. to carboxyl groups, requires much bigger doses than one would use for normal data collection, but nowadays that may be not be true. It would be interesting to know.Jon CooperOn 28 Apr 2020 16:37, "Thomas, Leonard M."  wrote:
Hello all,


This is one of those issues that seems to come up now and then.  I have been working on a structure that obviously has some radiation damage as indicted by negative density and/or high thermal parameters.  Since we know that residue X is in the
 sequence the sidechain should be there and is just flopping around or has been damaged/removed by radiation exposure.  My questions is what is the current thinking on presenting these residues for deposition.  Remove the side chain atoms, drop the occupancy
 to zero, just let them  behave as they want ie high B factors some negative density.  


Cheers,
Len





Leonard Thomas

lmthomas@ou.edu










To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] Methods to improve ligand density of a homodimer?

[00000c2488af9525-dmarc-request]

I take it that you have 2 molecules in the asymmetric unit. If so, you could try some sort of NCS averaging of the map or just NCS-refinement might help. Not much more here than has been expertly suggested already. Can you give us an idea of the resolution? How many RMS have you contoured the map at?Jon Cooper (pre-polder person)On 20 Apr 2020 20:23, Kyle Gregory <3632e92fcc15-dmarc-requ...@jiscmail.ac.uk> wrote:

Hello all, 





I have a homodimer structure in P1 21 1 spacegroup, the dimer is likely a crystallographic artefact, where it looks like the monomer is rotated by ~ 180 degrees around the Y axis.


I am assessing ligand binding and each of the monomers display density at the site but it is not as clear as I would like. A polder map does help things but I was wondering if it is feasible, or if there are any tools, that can be used to improve density based
 of the fact there are two molecles present. Is there some way to sum (probably the wrong word here) the densities at the binding site?

Kind regards,

Kyle 






To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] molecular graphics program accessibility

[00000c2488af9525-dmarc-request]

There is CueMol which runs on iPhone. NDKmol/ESmol for Android. It's a few years since I used them, but they were good for teaching purposes. As suggested though, a web-based viewer is probably the way to go. Jon CooperOn 17 Apr 2020 15:43, "careinaedgo...@yahoo.com" <02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:Dear all,I have to teach online this year. I usually ask students to view and study protein structures using SPDBV and pymol. Does anyone know if these programs would run on smart devices? Not all students have computers. Or are there any alternative viewing programs that run on smart devices? thank you, Careina


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] CCP4i2 Text size

[00000c2488af9525-dmarc-request]

I had similar thing with linux (not Windows) and the cure was (from memory) to install the Truetype font. Absolutely no idea if this is relevant in your case!!Jon CooperOn 2 Apr 2020 14:51, "Horrell, Sam (DLSLtd,RAL,LSCI)"  wrote:

Hi Huw, 
 
Thanks for the advice, but I’m afraid my display scaling is actually at 250%. I’ve changed the setting you suggested but it hasn’t changed much.

 
Sounds like a bug fix is on the way for the scaling problem though.

 
Cheers, 
 
Sam 


From: Huw Jenkins 

Sent: 02 April 2020 14:04
To: Horrell, Sam (DLSLtd,RAL,LSCI) 
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] CCP4i2 Text size


 
Hi Sam,




On 2 Apr 2020, at 11:32, Horrell, Sam (DLSLtd,RAL,LSCI)  wrote:

 If anyone has any other suggestions it would be greatly appreciated.

 

What settings do you have for Windows display scaling? From your screenshot it appears that the Windows task bar and window title bar are drawn at >100%


 


If so and your whole desktop is scaled can you try turning off "Let Windows try to fix apps so they're not blurry" in the Advanced scaling settings please? Then restart CCP4i2 and set the font sizes back to more normal sizes. This will
 make the text look less crisp but should make CCP4i2 more usable. 


 


Best wishes,


 


 


Huw 


 





 
-- 
This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail.Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message.Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom 



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] Overrefinement considerations and Refmac5.

[00000c2488af9525-dmarc-request]

Well, as no one else has come back to you, I wouldn't say 10 % difference between R and R-free is 'bad' and it's certainly not super-bad! It's a bit high, but if you look at the papers by Ian Tickle from the late 90's and others I think you can be reasonably happy with it. I've been told that A N other well-known protein crystallography suite gives a lower difference for the same structure, so you could always try that ;-)Jon CooperOn 6 Mar 2020 13:36, M T  wrote:Dear BBers,I am trying to refine a structure using COOT and Refmac5 and I have some concerns about overrefinement and x-ray term weight in Refmac5, based on the fact that during refinement to let R factor to drift too far from Rfree is not good...So... First question about that : what is too far ? I have some values in mind like 6% of difference is OK, 10% is not... But is there a relation in between resolution of the structure and this difference? Should it be higher at lower resolution, or always around 6-7% independently of the resolution?Second question is, ok, I have a too big difference, lets say 9-10%... What could be the reason of that and on what to play to reduce this difference?One way I choose is to look at the x-ray term weight (even if I am totally sure that Refmac5 is doing things better than me), because I saw that the final rms on BondLength were to constraint (I have in mind that this value should stays in between 0.02 and 0.01).So I looked into Refmac log to know where was the starting point and I found 8.75.Then I tried several tests  and here are the results: 

*
  
  R factor
  
  
  Rfree
  BondLength
  
  BondAngle
  
  
  ChirVolume
  
  Auto weighting and experimental sigmas boxes checked
  
  0.1932
  0.2886
  
  0.0072
  
  1.6426
  
  
  0.1184
  
  Weighting term at 4 and experimental sigmas box checked
  
  0.1780
  0.3159
  
  0.1047
  
  8.1929
  
  
  0.5937
  
  Weighting term at 4
  
  0.1792
  0.3143
  
  0.1008
  
  7.8200
  
  
  0.5667
  
  Weighting term at 15 and experimental sigmas box checked
  
  0.1783
  0.3272
  
  0.2020
  
  1.6569
  
  
  0.9745
  
  Weighting term at 15
  
  0.1801
  0.3279
  
  0.2022
  
  12.5748
  
  
  0.9792
  
  Weighting term at 8.75
  
  0.1790
  0.3235
  
  0.1545
  
  10.5118
  
  
  0.7909
  
  Auto weighting box checked
  
  0.1948
  0.2880
  
  0.0076
  
  1.6308
  
  
  0.1176
  

 Refinement ParametersSo like nothing looks satisfying I decided to ask my questions here...What do you recommend to fix my problem, which is a too large difference between R and Rfree?Thank you for answers.








To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] Hydrogens in PDB File

[00000c2488af9525-dmarc-request]

I was going to say there's no harm in depositing hydrogen atom positions! However, some structures of similar resolution that we refined with shelx using riding H's and anisotropic B's, somewhat embarrassingly, do not seem to have them! These were deposited about 20 years ago so my memory is vague. Maybe there is or was some restriction on depositing H-atom coordinates, or more probably we found that having them in the PDB file (in the pre-Coot era) was guaranteed to crash the average graphics program of the day or at least scramble the displayed structure for whoever downloaded the coordinates. I suspect the latter.Jon CooperOn 28 Feb 2020 00:07, "Whitley, Matthew J"  wrote:

Hi Ethan, thanks for your reply.  The correct situation is the former: hydrogens added at idealized positions *before* refinement and then subjected to refinement along
 with the rest of the model.
 
After refinement, MolProbity (the online server) does indeed remove any hydrogens in the PDB file and add them back at idealized positions for the purpose of its calculations,
 but I am most definitely *not* talking about depositing these post-refinement hydrogens manipulated by MolProbity.

 
Matthew
 
---
Matthew J. Whitley, Ph.D.
Research Instructor
Department of Pharmacology & Chemical Biology
University of Pittsburgh School of Medicine
 

From: Ethan A Merritt
Sent: Thursday, February 27, 2020 6:57 PM
To: Whitley, Matthew J
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] Hydrogens in PDB File

 
On Thursday, 27 February 2020 15:35:05 PST Whitley, Matthew J wrote:
> Hello all,
> 
> I am nearly finished refining the structures of two mutant proteins from
> crystals that diffracted to very high resolution, 1 Å and 1.2 Å,
> respectively.  Refinement was conducted in the presence of explicit
> hydrogens on the models.  I am preparing to deposit these models into the
> PDB but am unsure about whether to retain or remove the hydrogens for
> deposition.  On one hand, these hydrogens were explicitly used during
> refinement, so that makes me want to keep them, but on the other hand, they
> were added at theoretical positions by MolProbity’s reduce tool for
> refinement and were not positioned on the basis of experimentally observed
> electron density, so that makes me want to delete them from the
> experimental model.  Which is the preferred option for this situation?

The order of operations you describe is unclear.

If you explicitly refined hydrogens then their final positions are indeed
based on experimentally determined data.
The fact that you initially placed them into ideal geometry is not really
any different from the non-H atoms of individual protein residues in your
model, whose original positions were also based on known stereochemistry.

On the other hand, if you mean that the hydrogens you used for refinement
were deleted and replaced during validation by Molprobity (which I think it
may do by default) that's not good.  You should rather keep the hydrogen
positions from refinement, not the ones from Molprobity.

Assuming (since this is ccp4bb) you refined with refmac...
- If you are at the level of investigating hydrogen positions, you may want
to consider taking the refinement into shelxl.  
- If you are not making claims about hydrogens but just want to describe
what you did during refinement, I'd go with taking them out and settling
for the standard record in the resulting PDB file:
  REMARK   3  HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
which looks like this in the corresponding mmcif file:
  _refine.details   'Hydrogens have been added in their riding positions'

    Ethan

> 
> Thanks,
> Matthew
> 
> ---
> Matthew J. Whitley, Ph.D.
> Research Instructor
> Department of Pharmacology & Chemical Biology
> University of Pittsburgh School of Medicine
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> 
https://nam05.safelinks.protection.outlook.com/?url="">


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


 




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] Shipping samples for neutron diffraction

[00000c2488af9525-dmarc-request]

... and don't say you're travelling with heavy water!Jon CooperOn 19 Feb 2020 17:12, "Azadmanesh, Jahaun"  wrote:

Hello,




I have traveled to a neutron beamline ~6 times over the past several years.




I have had awful luck shipping crystals, so I decided to hand-carry and I found this best.




I'm not sure what conditions you plan to shoot your crystals.  I mount my crystals in a sturdy quartz capillary (from vitrocom), seal the ends with a layer of wax, then two coats of nail polish (choose your favorite prettiest color!) 




I would then wrap the capillary in cotton from a cottonball, then "stuff" the capillary-cotton combination in a 15 mL falcon tube for a snug fit. For extra assurance, the falcon tubes were placed inside a generous amount of bubble-wrap that was then taped inside
 a small cardboard box. 




I have sought anaerobic conditions for several of my samples to acquire a desired redox state of my metallo-protein. This was tough, so instead I used a high-ish concentration of my redox agent dissolved in my reservoir solution "slugs" in the capillary. These
 would be replaced in intervals with fresh slugs prior to the beamtime, and for sure right before the beamtime. The redox state held for my ~10 days of beamtime and several months after.




For cryo-conditions, I just placed a bunch of my crystals in a capillary filled with reservoir solution and sealed as above. I would do my chemical manipulations and freezing at the beamline.




Much more details are found in our publication: https://www.ncbi.nlm.nih.gov/pubmed/30279321 




Hope this helps!


From: CCP4 bulletin board  on behalf of stephen.c...@rc-harwell.ac.uk 
Sent: Wednesday, February 19, 2020 4:22 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Shipping samples for neutron diffraction
 


Non-UNMC email 

Dear CCP4 community,


I have an impending trip to a neutron source and was wondering how people tend to ship their samples prior to beam time?  Is sending something frozen best or is sealed in a capillary more sensible or is there another better way?  One caveat is that ideally
 my sample should remain anaerobic which has me leaning towards frozen, but I guess in pcr type tubes sealed in gas tight vessels is also a viable option?


Any thoughts or suggestions are very much appreciated.


Best wishes,


Steve


Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717

This email and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorized recipient of the addressee, please notify us of receipt
 by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to this email.

Any views or opinions presented are solely those of the author and do not necessarily represent those of the Research Complex at Harwell.

There is no guarantee that this email or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message.

We use an electronic filing system. Please send electronic versions of documents, unless paper is specifically requested.

This email may have a protective marking, for an explanation, please see:
https://urldefense.com/v3/__http://www.mrc.ac.uk/About/informationandstandards/documentmarking/index.htm__;!!JkUDQA!b5-sOFziDpUSOnWUVY3BWn_6eloBp1th-M3qGa2sBfF1-Ak7AgXiHT91jdMOlwo3yU5MtpQ$
 .




To unsubscribe from the CCP4BB list, click the following link:
https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1__;!!JkUDQA!b5-sOFziDpUSOnWUVY3BWn_6eloBp1th-M3qGa2sBfF1-Ak7AgXiHT91jdMOlwo37r205do$




The information in this e-mail may be privileged and confidential, intended only for the use of the addressee(s) above. Any unauthorized use or disclosure of this information is prohibited. If you have received this e-mail by mistake, please delete it and immediately
 contact the sender.



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] Lattice-translocation defect (LTD)

[00000c2488af9525-dmarc-request]

I was wondering why you believe it is a lattice defect rather than NCS. How well does the structure refine and does it have NCS? Do the twinning tests suggest anything?Jon CooperOn 11 Feb 2020 21:31, Daniele de Sanctis  wrote:Hi all,I'm currently dealing with what I think it is a case of LTD (off-origin Patterson peak, with vector along w of ~ 7A and electron density map showing a "ghost" map shifted by 7 A). I saw there are quite a few cases reported in literature  (for example Hare et al, 2006), but what I could not find is how I can demodulate the data. Is there any software that can be used for this?Thank youDaniele-- ἀρετή---Daniele de Sanctis, PhDStructural Biology GroupESRF, Grenoble, FranceTel 33 (0)4 76 88 2869


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] protein protein interactions

[00000c2488af9525-dmarc-request]

Hello, if you mean theoretical docking, here is an old list of links:https://zlab.umassmed.edu/zdock/dockingsites.shtmlSome of these will still be maintained.Jon CooperOn 10 Feb 2020 16:50, Sarah Bowman  wrote:

Hi Careina,
 
There’s a program called FRODOCK that generates predictions of how two proteins could interact:
http://chaconlab.org/modeling/frodock
 
Cheers,
Sarah
 

Sarah EJ Bowman, PhD
 
Associate Research Scientist, Hauptman-Woodward Medical Research Institute
Director, High-Throughput Crystallization Screening Center
 
Research Webpage
www.getacrystal.org


 
 

From: CCP4 bulletin board  on behalf of "careinaedgooms@yahoo.com" <02531c126adf-dmarc-request@JISCMAIL.AC.UK>
Reply-To: "careinaedgooms@yahoo.com" 
Date: Monday, February 10, 2020 at 11:17 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Fw: protein protein interactions


 



 










 









Dear all


Apologies for off topic question but can anyone recommend good programs for identifying docking interfaces between two proteins. I do not know that these two proteins
 interact. I would like a level of confidence on a possible interaction. is there a good program to do this?


kind regards


Careina













 



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1





To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] refinement of 0.73A data in shelxl

[00000c2488af9525-dmarc-request]

the swapping of the lines and I did not understand Tim's comment "The scattering factor is derived from the number next to the name."
Once I adjusted the numbers in the second column of my inhibitors to match the DISP list numbering, Rfree dropped to 16.96% and the map looks notably better (see attached snap shot).



Again, thank you very much for such an incredible feedback.
Best, Matthias










Dr. Matthias Barone
AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284





From: CCP4 bulletin board  on behalf of Tim Gruene 
Sent: Tuesday, February 4, 2020 9:24:24 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl
 



Dear Jon,

in SHELX(L), you can name your atoms foo and bar, or jon and doe, if you like. 
The scattering factor is derived from the number next to the name. The name is 
just that, and identifier.

Best,
Tim

On Monday, February 3, 2020 9:20:03 PM CET 0c2488af9525-dmarc-
request@JISCMAIL.AC.UK wrote:
> Remembered earlier that if the "CL" is not shifted one place to the left,
> Shelx and probably most other programs treat it as carbon, i.e. its assumed
> to have 6 rather than 17 electrons. Trust occupancies OK, too ;-?
> 
> 
> Jon Cooper
> 
> 
> On 3 Feb 2020 18:26, "Barone, Matthias"  wrote:
> 
> 
> Hi Pavel
> 
> glad you write me. I was hoping you would read my post.
> 
> - Yes, protons are added, both on the protein as well as on the molecule
> 
> - I initially only refined protein and ligand anisotropically, now Im
> running a refinement with all atoms anisotrp except Hs. This would then
> also be the same as shelxl is doing.
> 
> - Alternate conformations are modeled, also on the ligand. There are plenty,
> sure, but I think I got most of them.
> 
> - I already used Water update during refine, there are some NO3s in the
> structure. I got them in. There is a second ligand somewhere as artifact.
> its density is not well defined, so I hope to get that in once the map
> clears up more. 
> 
> - I let phenix.refine optimize adp and chemisty weights, but as Petri
> suggested, Im manually increasing the scale factors to match the ones from
> shelxl (just to compare them properly). Im aiming for an rsmd of 0.02-0.03A
> like Petri suggested and keep an eye on how tight the structure is refined
> in shelxl.
> 
> 
> 
> 
> About the Rfact and the gap. Yes, thats what I was expecting. I hope if I
> add more anisotropic B fact, the Rfacts should go down to at least what
> shelxl yielded. 
> 
> 
> 
> 
> thank you all again for the massive feedback, ideas and help. 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> Dr. Matthias Barone
> 
> AG Kuehne, Rational Drug Design
> 
> 
> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
> Robert-Rössle-Strasse 10
> 13125 Berlin
> 
> Germany
> Phone: +49 (0)30 94793-284
> 
> 
> From:Pavel Afonine 
> Sent:Monday, February 3, 2020 7:14:25 PM
> To:Barone, Matthias
> Cc:CCP4BB@JISCMAIL.AC.UK
> Subject:Re: [ccp4bb] refinement of 0.73A data in shelxl
>  
> Hi Matthias,
> 
> 
> did you use correct model parameterization and optimal refinement strategy
> for the resolution? Such as: - Add H atoms;
> - Refine all but H atoms with anisotropic ADPs;
> - Model alternative conformations (that one'd expect many at this
> resolution); - Add solvent (water, crystallization cocktail components if
> you see any); - Relax restraints on geometry and ADPs;
>  long list!
> 
> 
> If not, then what you have in terms of R factors is more or less what I'd
> expect.
> 
> 
> In the absence of obvious data pathologies, I'd expect Rwork/Rfree in 10-15%
> range, and the Rfree-Rwork gap around 1-2% or less.
> 
> 
> Since you mentioned Phenix refinement, I am happy to help you with details
> etc off-list.
> 
> 
> Pavel
> 
> 
> On Mon, Feb 3, 2020 at 3:08 AM Barone, Matthias 
> wrote:
> 
> 
> Dear ccp4 community
> 
> Im having some problems solving a 0.73A structure. Spacegroup seems to be
> correct, data are not twinned, 95.5% overall completeness, ISa 25.6. Outer
> shell CC1/2 24% and 90.4% complete.
> 
> The model is nearly fully built, there is no remaining unmodelled areas.
> However, Rfactisstuck 27% in phenix, with a very distinct artifact in the
> electron map (see phenix.jpg). You can see difference density on various
> well defined sidechain atoms. Notably, they seem to follow a p

Re: [ccp4bb] refinement of 0.73A data in shelxl

[00000c2488af9525-dmarc-request]

Hello, hope I can help.OK, so here is the disp table...
SFAC  C H CL N O  
DISP $C     0.00510    0.00239     15.73708
DISP $H    -0.2    0.0      0.66954
DISP $CL    0.18845    0.21747   1035.16450
DISP $N     0.00954    0.00480     28.16118
DISP $O     0.01605    0.00875     47.79242If we take these coordinates...
N     3    0.414964   -0.147635    0.116896    11.0    0.19533    0.44341 =
H0A   2    0.427823   -0.138656    0.123256    11.0   -1.5
C     1    0.348035   -0.160776    0.110979    11.0    0.20723    0.28451 =
O     4    0.363785   -0.174154    0.102906    11.0    0.21226    0.22954 =
SG    5    0.177303    0.101267    0.040572    10.04000    0.06849    0.03024 =
O     4    0.241304    0.071735    0.038567    10.96000    0.14982    0.12755 =


... the first N (followed by 3) is being assigned the scattering factors of chlorine because this element is 3rd in the SFAC list. The SG (followed by 5) is being assigned the scattering factors of O because the latter is 5th in the SFAC list.
I think you need to check these  assignments and the chlorine occupancy are Ok.Jon CooperOn 6 Feb 2020 11:13, "Barone, Matthias"  wrote:





Dear community
here is an update of my shelxl problem. I solved it after an epiphany last night in bed...
I tried countless things to get the postive density on the Cl under control. 
Markus suggested that the density came from a radiolysed chloride, so I tried to superimpose chlorinated and radiolysed ligands.
However that did not lead to anything fruitful. 


Remember that I tried to incorporate DISP of Cl into the .ins file:
This is the original of the protein .ins, chloride just pasted as last element:
SFAC  C  H  N  O  S  CL
DISP $C     0.00510    0.00239     15.73708
DISP $H    -0.2    0.0      0.66954
DISP $N     0.00954    0.00480     28.16118
DISP $O     0.01605    0.00875     47.79242
DISP $S     0.15995    0.16998    812.87489
DISP $CL    0.18845    0.21747   1035.16450


The upper list only creates postive density on the Chloride, the rest of the map is clean and looks the same as if you would omit the DISP line of Cl alltogether.
The following list is coming from the .ins file of the converted prodrg file:


SFAC  C H CL N O  
DISP $C     0.00510    0.00239     15.73708
DISP $H    -0.2    0.0      0.66954
DISP $CL    0.18845    0.21747   1035.16450
DISP $N     0.00954    0.00480     28.16118
DISP $O     0.01605    0.00875     47.79242
UNIT  38 48 1 5 7  


Pasting CL as third element in the .ins file, however, created these weird difference signals on the backbone O and N that I mentioned. 
You can probably see where this is going. Here are some atoms of the protein in the .ins file:


N     3    0.414964   -0.147635    0.116896    11.0    0.19533    0.44341 =
H0A   2    0.427823   -0.138656    0.123256    11.0   -1.5
C     1    0.348035   -0.160776    0.110979    11.0    0.20723    0.28451 =
O     4    0.363785   -0.174154    0.102906    11.0    0.21226    0.22954 =
SG    5    0.177303    0.101267    0.040572    10.04000    0.06849    0.03024 =
O     4    0.241304    0.071735    0.038567    10.96000    0.14982    0.12755 =


And here are some atoms of the inhibitor:


OBM   5    0.325170    0.441790    0.181777    11.0    0.42576    0.30731 =  <- oxygen
CE1   1   -0.036497    0.262177    0.187030    11.0    0.12056    0.22455 =  <- carbon
HE1   2   -0.028898    0.247344    0.187663    11.0   -1.2
<- proton
NAY   4    0.107745    0.387704    0.210972    11.0    0.16719    0.14264 = <- nitrogen
CLAA   3  0.028744999  0.27121   0.199305996 0.5   
<- Chloride


Turned out that Jon had a good feeling about the swapping of the lines and I did not understand Tim's comment "The scattering factor is derived from the number next to the name."
Once I adjusted the numbers in the second column of my inhibitors to match the DISP list numbering, Rfree dropped to 16.96% and the map looks notably better (see attached snap shot).



Again, thank you very much for such an incredible feedback.
Best, Matthias










Dr. Matthias Barone
AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284





From: CCP4 bulletin board  on behalf of Tim Gruene 
Sent: Tuesday, February 4, 2020 9:24:24 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl
 



Dear Jon,

in SHELX(L), you can name your atoms foo and bar, or jon and doe, if you like. 
The scattering factor is derived from the number next to the name. The name is 
just that, and identifier.

Best,
Tim

On Monday, February 3, 2020 9:20:03 PM CET 0c2488af9525-dmarc-
request@JISCMAIL.AC.UK wrote:
> Remembered earlier that if the "CL&quo

Re: [ccp4bb] refinement of 0.73A data in shelxl

[00000c2488af9525-dmarc-request]

Thanks, sorry, shelx is wonderful! I was thinking back to shelxpro days when your hetero-atoms could acquire the wrong scattering factors if they were not positioned right in the pdb file. The classic, which I saw several times, was calcium (CA) being treated as an alpha-carbon (CA), and I think refmac and others probably still do this if the PDB-standard leftward offset of the atom type is not present.Jon CooperOn 4 Feb 2020 08:24, Tim Gruene  wrote:Dear Jon,

in SHELX(L), you can name your atoms foo and bar, or jon and doe, if you like. 
The scattering factor is derived from the number next to the name. The name is 
just that, and identifier.

Best,
Tim

On Monday, February 3, 2020 9:20:03 PM CET 0c2488af9525-dmarc-
requ...@jiscmail.ac.uk wrote:
> Remembered earlier that if the "CL" is not shifted one place to the left,
> Shelx and probably most other programs treat it as carbon, i.e. its assumed
> to have 6 rather than 17 electrons. Trust occupancies OK, too ;-?
> 
> 
> Jon Cooper
> 
> 
> On 3 Feb 2020 18:26, "Barone, Matthias"  wrote:
> 
> 
> Hi Pavel
> 
> glad you write me. I was hoping you would read my post.
> 
> - Yes, protons are added, both on the protein as well as on the molecule
> 
> - I initially only refined protein and ligand anisotropically, now Im
> running a refinement with all atoms anisotrp except Hs. This would then
> also be the same as shelxl is doing.
> 
> - Alternate conformations are modeled, also on the ligand. There are plenty,
> sure, but I think I got most of them.
> 
> - I already used Water update during refine, there are some NO3s in the
> structure. I got them in. There is a second ligand somewhere as artifact.
> its density is not well defined, so I hope to get that in once the map
> clears up more. 
> 
> - I let phenix.refine optimize adp and chemisty weights, but as Petri
> suggested, Im manually increasing the scale factors to match the ones from
> shelxl (just to compare them properly). Im aiming for an rsmd of 0.02-0.03A
> like Petri suggested and keep an eye on how tight the structure is refined
> in shelxl.
> 
> 
> 
> 
> About the Rfact and the gap. Yes, thats what I was expecting. I hope if I
> add more anisotropic B fact, the Rfacts should go down to at least what
> shelxl yielded. 
> 
> 
> 
> 
> thank you all again for the massive feedback, ideas and help. 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> Dr. Matthias Barone
> 
> AG Kuehne, Rational Drug Design
> 
> 
> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
> Robert-Rössle-Strasse 10
> 13125 Berlin
> 
> Germany
> Phone: +49 (0)30 94793-284
> 
> 
> From:Pavel Afonine 
> Sent:Monday, February 3, 2020 7:14:25 PM
> To:Barone, Matthias
> Cc:CCP4BB@JISCMAIL.AC.UK
> Subject:Re: [ccp4bb] refinement of 0.73A data in shelxl
>  
> Hi Matthias,
> 
> 
> did you use correct model parameterization and optimal refinement strategy
> for the resolution? Such as: - Add H atoms;
> - Refine all but H atoms with anisotropic ADPs;
> - Model alternative conformations (that one'd expect many at this
> resolution); - Add solvent (water, crystallization cocktail components if
> you see any); - Relax restraints on geometry and ADPs;
>  long list!
> 
> 
> If not, then what you have in terms of R factors is more or less what I'd
> expect.
> 
> 
> In the absence of obvious data pathologies, I'd expect Rwork/Rfree in 10-15%
> range, and the Rfree-Rwork gap around 1-2% or less.
> 
> 
> Since you mentioned Phenix refinement, I am happy to help you with details
> etc off-list.
> 
> 
> Pavel
> 
> 
> On Mon, Feb 3, 2020 at 3:08 AM Barone, Matthias 
> wrote:
> 
> 
> Dear ccp4 community
> 
> Im having some problems solving a 0.73A structure. Spacegroup seems to be
> correct, data are not twinned, 95.5% overall completeness, ISa 25.6. Outer
> shell CC1/2 24% and 90.4% complete.
> 
> The model is nearly fully built, there is no remaining unmodelled areas.
> However, Rfactisstuck 27% in phenix, with a very distinct artifact in the
> electron map (see phenix.jpg). You can see difference density on various
> well defined sidechain atoms. Notably, they seem to follow a pattern:
> Nearly all Val CG have difference signal, as well as many backbone
> NH. Hence, I suspected that it might be a problem with the SF, since we
> recorded the DS at 0.86A.
> 
> 
> 
> 
> Hence I gave shelxl a shot:
> 
> I used the refined model from phenix, converted it via pdb2ins and pasted
> the restraints created by prodrg. 
> 
> The shelxl hkl was produced by xdsconv, using the freeR flagging of the mtz
> used by phenix (no merge, friedel false).
> 
> Interestingly, shelxl can bring Rfree down to 16% and almost all of
> the diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg).
> Except one: the inhibitor contains a chlorinated phenylring (pdb ligand
> 2L5) which now shows massive difference density for Cl.
>

Re: [ccp4bb] refinement of 0.73A data in shelxl

[00000c2488af9525-dmarc-request]

Remembered earlier that if the "CL" is not shifted one place to the left, Shelx and probably most other programs treat it as carbon, i.e. its assumed to have 6 rather than 17 electrons. Trust occupancies OK, too ;-?Jon CooperOn 3 Feb 2020 18:26, "Barone, Matthias"  wrote:


Hi Pavel
glad you write me. I was hoping you would read my post.
- Yes, protons are added, both on the protein as well as on the molecule
- I initially only refined protein and ligand anisotropically, now Im running a refinement with all atoms anisotrp except Hs. This would then also be the same as shelxl is doing.
- Alternate conformations are modeled, also on the ligand. There are plenty, sure, but I think I got most of them.
- I already used Water update during refine, there are some NO3s in the structure. I got them in. There is a second ligand somewhere as artifact. its density is not well defined, so I hope to get that in once the map clears up more. 
- I let phenix.refine optimize adp and chemisty weights, but as Petri suggested, Im manually increasing the scale factors to match the ones from shelxl (just to compare them properly). Im aiming for an rsmd of 0.02-0.03A like Petri suggested and keep an
 eye on how tight the structure is refined in shelxl.


About the Rfact and the gap. Yes, thats what I was expecting. I hope if I add more anisotropic B fact, the Rfacts should go down to at least what shelxl yielded. 


thank you all again for the massive feedback, ideas and help. 








Dr. Matthias Barone
AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284





From: Pavel Afonine 
Sent: Monday, February 3, 2020 7:14:25 PM
To: Barone, Matthias
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl
 



Hi Matthias,


did you use correct model parameterization and optimal refinement strategy for the resolution? Such as:
- Add H atoms;
- Refine all but H atoms with anisotropic ADPs;
- Model alternative conformations (that one'd expect many at this resolution);
- Add solvent (water, crystallization cocktail components if you see any);
- Relax restraints on geometry and ADPs;
 long list!


If not, then what you have in terms of R factors is more or less what I'd expect.


In the absence of obvious data pathologies, I'd expect Rwork/Rfree in 10-15% range, and the Rfree-Rwork gap around 1-2% or less.


Since you mentioned Phenix refinement, I am happy to help you with details etc off-list.


Pavel


On Mon, Feb 3, 2020 at 3:08 AM Barone, Matthias  wrote:




Dear ccp4 community
Im having some problems solving a 0.73A structure. Spacegroup seems to be correct,
 data are not twinned, 95.5% overall completeness, ISa 25.6. Outer shell CC1/2
 24% and 90.4% complete.
The model is nearly fully built, there is no remaining unmodelled areas. However, Rfact
is stuck 27% in phenix, with a very distinct artifact in the electron map (see phenix.jpg). You can see difference
 density on various well defined sidechain atoms. Notably, they seem to follow a pattern: Nearly all Val CG have difference signal, as well as many backbone NH. Hence, I suspected that it might be a problem with the SF, since
 we recorded the DS at 0.86A. 



Hence I gave shelxl a shot:
I used the refined model from phenix, converted it via pdb2ins and pasted the restraints created by prodrg. 
The shelxl hkl was produced by xdsconv, using the freeR flagging of the mtz used by phenix (no merge, friedel false).
Interestingly, shelxl can bring Rfree down to 16% and almost all of the diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg). Except one: the inhibitor contains a chlorinated phenylring (pdb
 ligand 2L5) which now shows massive difference density for Cl. 
I therefore suggested that I might deal with a wrong SF for Cl. Funny enough, pdb2ins does not produce a DISP line for Cl if converting the pdb that contains the inhibitor. Hence,
I used pdb2ins and the pdb from PRODRG to produce SFAC for the inhibitor Cloride. I then pasted this line

DISP $CL    0.18845    0.21747   1035.16450

into the .res file and updated the UNIT line. Shelxl runs through, and the density looks ok on the Chloride now. However Rfree is back
 up at 24% and the artifacts seen by phenix.refine are back (shelxl_SFAC-CL.jpg): now, very distincitvly, backbone carbonyls and NHs show difference
 density.

Am I right in my assumption, that the SFAC of Cloride is not properly calculated at the given wavelenght? And if so, how do I guess it correctly?


Thank you very much for your help!
Best, matthias








Dr. Matthias Barone
AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284







To u

Re: [ccp4bb] refinement of 0.73A data in shelxl

[00000c2488af9525-dmarc-request]

Hello, sorry if this is a really obvious question, but have you used the ANIS option? I remember it is good at cleaning-up difference density around halogen atoms that sort of resolution.Jon CooperOn 3 Feb 2020 11:08, "Barone, Matthias"  wrote:

Dear ccp4 community
Im having some problems solving a 0.73A structure. Spacegroup
 seems to be correct, data are not twinned, 95.5% overall completeness, ISa 25.6. Outer shell CC1/2
 24% and 90.4% complete.
The model is nearly fully built, there is no remaining unmodelled
 areas. However, Rfact is stuck 27% in phenix, with a very distinct artifact in the electron map (see phenix.jpg).
 You can see difference density on various well defined sidechain atoms. Notably, they seem to follow a pattern: Nearly all Val CG have difference signal, as well as many backbone NH. Hence, I suspected that it might be
 a problem with the SF, since we recorded the DS at 0.86A. 



Hence I gave shelxl a shot:
I used the refined model from phenix, converted it via pdb2ins and pasted the restraints created by prodrg. 
The shelxl hkl was produced by xdsconv, using the freeR flagging of the mtz used by phenix (no merge, friedel false).
Interestingly, shelxl can bring Rfree down to 16% and almost all of the diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg). Except one: the inhibitor contains a chlorinated phenylring
 (pdb ligand 2L5) which now shows massive difference density for Cl. 
I therefore suggested that I might deal with a wrong SF for Cl. Funny enough, pdb2ins does not produce a DISP line for Cl if converting the pdb that contains the inhibitor. Hence,
I used pdb2ins and the pdb from PRODRG to produce SFAC for the inhibitor Cloride. I then pasted this line

DISP $CL    0.18845    0.21747   1035.16450

into the .res file and updated the UNIT line. Shelxl runs through, and the density looks ok on the Chloride now. However Rfree
 is back up at 24% and the artifacts seen by phenix.refine are back (shelxl_SFAC-CL.jpg): now, very distincitvly, backbone carbonyls and
 NHs show difference density.

Am I right in my assumption, that the SFAC of Cloride is not properly calculated at the given wavelenght? And if so, how do I guess it correctly?


Thank you very much for your help!
Best, matthias








Dr. Matthias Barone
AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284







To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] Protein fold and the moonlighting function

[00000c2488af9525-dmarc-request]

Can you possibly expand a bit on exactly what you are asking?Jon CooperOn 2 Feb 2020 09:28, Rajnandani Kashyap  wrote:Dear AllI am curious to know about the promiscuous activity of a protein based on their fold. Can a protein having same fold also have same function (say not 100% but some activity) as the homologous structure. Please also let me know few relevant PDB structures where such kind of activity is shown promising. RegardsRajnandani KashyapPhD Student


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

On 2 Feb 2020 09:28, Rajnandani Kashyap  wrote:Dear AllI am curious to know about the promiscuous activity of a protein based on their fold. Can a protein having same fold also have same function (say not 100% but some activity) as the homologous structure. Please also let me know few relevant PDB structures where such kind of activity is shown promising. RegardsRajnandani KashyapPhD Student


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

On 2 Feb 2020 09:28, Rajnandani Kashyap  wrote:Dear AllI am curious to know about the promiscuous activity of a protein based on their fold. Can a protein having same fold also have same function (say not 100% but some activity) as the homologous structure. Please also let me know few relevant PDB structures where such kind of activity is shown promising. RegardsRajnandani KashyapPhD Student


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] AW: [ccp4bb] Urea vs. Guanidinium/HCl

[00000c2488af9525-dmarc-request]

Hello, have you tried eluting from nickel-NTA straight into glycerol buffer so the protein has absolutely minimal time on it's own? Also, 10 mg/ml is quite a high concentration in my book. What is in your purification buffer? Just interested. Hope your competitors aren't reading all this! Best wishes.Jon CooperOn 2 Feb 2020 12:13, John Newitt  wrote:Have you tried maintaining the protein in a solution with a low concentration of excess cofactor? Light vs dark? Excess reducing agent or different reducing agent or oxygen-depleted and under argon?JohnOn Feb 2, 2020, at 4:22 AM, Jon Hughes  wrote:Thanks for you interest. Ok, here are some more details. The protein is Cph1 (uniprot Q55168) as a full-length (ca. 80 kDa) holophytochrome, produced with a C-terminal His6 tag together with its co-factor in E. coli, purified via NiNTA and SEC. It is red/far-red photochromic (that is, photoactive) such that, as a 2 component sensory histidine autokinase / phosphotransferase, its kinase activity can be switched on and off by appropriate light pulses. Thus it is unambiguously functional. It is also highly soluble (10 mg/ml is no problem) – but subsequently (over days and weeks) it aggregates (irrespective of the photostate) to form a fluffy precipitate. Incidentally, I believe that most SHPK's and indeed most phytochromes have aggregation problems like this. Beyond urea being a less potent chaotrope than guanidinium/HCl, the different chemical actions of the two might give a hint as to what causes the aggregation.Cheersjon Von: CCP4 bulletin board  Im Auftrag von Artem EvdokimovGesendet: Sonntag, 2. Februar 2020 00:46An: CCP4BB@JISCMAIL.AC.UKBetreff: Re: [ccp4bb] Urea vs. Guanidinium/HCl More details would be helpful. Do you know whether your protein is folded and active to begin with? Many partially folded proteins behave in a way that resembles your experience... Urea is a less potent denaturant mole for mole than GuHCl so it is not super surprising that it behaves differently. Artem On Sat, Feb 1, 2020, 6:22 PM Jon Hughes  wrote:Hello everyone,We work on a protein that tends to aggregate. The process is slowed but notstopped by glycerol and NDSB201. Interestingly, whereas guanidinium/HCldissolves the aggregate readily, urea just turns it into an amorphouschewing-gum-like mass. Does that info provide anyone with a clue as to whythe aggregation occurs and maybe suggest how to stop it in a way that wouldnot thwart crystal formation?Best,jon To unsubscribe from the CCP4BB list, click the following link:https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 To unsubscribe from the CCP4BB list, click the following link:https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] Unusual monomer-monomer interface in crystal

[00000c2488af9525-dmarc-request]

It looks pretty metallic. It would be good to know the max peak height in rms for the difference map and also some of the distances between the His and Cys side chains. Are your sulphur occupancies alright ;-?On 21 Jan 2020 17:55, Chris Fage  wrote:Dear CCP4BB Users,I've recently solved the ~2.2 angstrom structure of a protein. In my electron density there are unusual monomer-monomer interfaces involving pairs of His and Cys residues (see https://ibb.co/wdWBcdk). Note the positive Fo-Fc density between the four side chains. As there is not adequate space for a water molecule or metal ion, perhaps the Cys residues are partially tied up disulfide bonds? However, the protein looks to be fully monomeric based on LC-MS measurements. Has anyone else observed crystal-driven formation of disulfide bridges?Aside from this region, there is no extensive interface between momoners, and PDBePISA suggests a monomeric state.Thanks in advance for any advice!Best wishes,Chris


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] best program for merging the two datasets

[00000c2488af9525-dmarc-request]

I think Blend runs Pointless so that way you will get the best of both worlds.On 15 Jan 2020 14:08, Firdous Tarique  wrote:Hi.I have collected multiple datasets for my crystals and now want to merge them. Iwanted to know that between Blend and pointless, which programme is better to merge two or more data sets?ThanksKahkashan


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1

Re: [ccp4bb] Potential weak binding ligand in the active site

[00000c2488af9525-dmarc-request]

Hello, the statistics all look good apart from the R-merge and R-meas. It might be worth looking at the processing again in case it can be improved. I assume you mean rms rather than A when you say the difference density only disappears at 6 A and, if so, it must be a strong feature. Does the fragment you have refined make chemical sense, either as a possible hydrolysis product, impurity or distinct binding group? Sorry, not much helpful advice here.On 20 Dec 2019 04:57, Katherine Lim  wrote:Hi all,I apologise in advance for the long post. I am working on solving a structure that looks like it could have a ligand bound in the active site. My data was obtained from a crystal of just the soluble domain of my protein that had been soaked overnight in the ligand solution. The apo crystal structure is already known and so I have solved my structure using phaser MR. I have attached the Aimless report output of my structure at the end of this email. The current R values I have after refinement and adding in all the waters are Rfree: 0.2413 and Rwork: 0.1897.  I can clearly see green density that is much larger (only disappears when I contour the Fo-Fc map to about 6 A) than in my control crystal that had been soaked in the same concentration of just solvent (I had used DMF).I am struggling to add in my ligand as it doesn't seem like the entire ligand can fit in the green density. We think it may be because we have only used the soluble domain and so the ligand isn't held very securely since the transmembrane domain is missing. I have been trying to fit in smaller sections of it and doing an occupancy refinement. So far I have been able to get part of the ligand in with an occupancy of about 0.6 but after the refinement run, phenix.refine seems to move this part of the ligand slightly out of the area where I had tried to fit it into the green density. Interestingly, there isn't a big red density in the area that this ligand section has moved to. I would appreciate any advice on how I should proceed with trying to figure out if I have tried the correct section of the ligand and the kind of refinement settings to use with a weak binder. 



	
	
	
	
	
	



Space GroupP212121Unit cell abc84.76, 89.84, 91.55unit cell alpha beta gamma90, 90, 90OVERALLLOW RESHIGH RESLow res limit45.7845.781.94High res limit1.99.111.9Rmerge 0.2340.0521.684Rmeas0.2440.0541.768Rpim0.0690.0160.527Total # observations663073628236851Total # unique551745793359I/sigma7.527.91.4CC ½0.9970.9990.747Completeness %9998.794.7Multiplicity1210.811



Katherine Lim PhD CandidateSchool of Biomedical Sciences; School of Molecular Sciences    Marshall Centre for Infectious Disease Research and Training     The University of Western Australia    




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1