Re: [ccp4bb] the structures of Nucleic acid

[Phoebe A. Rice]

For complex RNA structures,

  *   Molecular replacement can stumble
  *   If made by simple T7 transcription methods (the most common way), the RNA 
would have EVERY U replaced with 5-bromo-U
  *   Adding bromine to the 5 position of U could affect the structure (it 
would be replacing -H not -CH3)

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


From: CCP4 bulletin board  on behalf of Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
Date: Monday, September 18, 2023 at 4:44 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] the structures of Nucleic acid
I am afraid most scientists will use the most straightforward technique!
If SAD is available the PHOSPHATE backbone of DNA will provide sufficient 
signal to allow SAD to work, and you get an unambiguous answer to whether it is 
A-DNA or B or Z...
MR will usually work of course as well
Eleanor


On Mon, 18 Sept 2023 at 09:18, Natesh Ramanathan 
mailto:nat...@iisertvm.ac.in>> wrote:
Dear Fu Xingke,

 Depends on what Nucleic Acid you are talking of.  If it is RNA, you 
can expect some sequence to tertiary structure correspondence so you might be 
able to try more MR as compared to DNA.   DNA may have double helical 
architecture but less sequence to tertiary structure correspondence, and hence 
DNA is less likely to have a 3D structure like RNA specific structure for a 
sequence.

 SAD has become a straight forward method to avoid all these problems 
to get ab-initio structure.  So many go for it directly.

Hope that helps.
Best wishes,
Natesh

On Mon, 18 Sept 2023 at 13:36, fuxingke 
mailto:fuxingke0...@163.com>> wrote:
Dear Colleagues,
 Reacently, I find the structures of Nucleic acid are solved by 
single-wavelength anomalous diffraction(SAD). So, why molecular replacement 
(MR) not?

Regards



Best wishes,
Fu Xingke
Institute of Physics CAS



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


--
--
"Live Simply and do Serious Things .. "
- Dorothy Mary Crowfoot Hodgkin OM, FRS

"In Science truth always wins"
- Max Ferdinand Perutz OM FRS
--
Dr. Ramanathan Natesh
Associate Professor,
School of Biology and Center for High-Performance Computing (CHPC),
Founding and Current President of Cryo Electron Microscopy and 3 Dimensional 
Image Processing Society of India (CEM3DIPSI),
Indian Institute of Science Education and Research Thiruvananthapuram 
(IISER-TVM),
Maruthamala P.O., Vithura,
Thiruvananthapuram,  695551, Kerala, India

nat...@iisertvm.ac.in
http://faculty.iisertvm.ac.in/natesh

Researcher ID: http://www.researcherid.com/rid/C-4488-2008
ORCID: http://orcid.org/-0002-1145-5962
Vidwan-ID : 94134: http://iisertvm.irins.org/profile/94134
PUBLONS: https://publons.com/author/1520837/ramanathan-natesh#profile

Office Ph. 0091- 471-2778087



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



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



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

Re: [ccp4bb] Intractable outliers in wwPDB validation report

[Nitin Kulhar]

Hello everyone.
Many thanks for the thoughts on the matter.

Dear Dr Abhinav

You can try to run a simulated annealing in CNS and then try to refine that
> model and see if that solves the problem. This would most likely help in
> getting rid of the overfitting problems.
>

I have trouble installing CNS on my machine which has Windows OS.
Nevertheless, rigid body refinement (RBR) and simulated annealing (SA) (and
combinations thereof) with phenix led to distant Rf and Rw (a sign of model
bias) at the beginning of refinement and even more so towards the end (*at
tedium*). Toggling off of RBR and SA, however, bridged the gap between Rf
and Rw while lowering them at the same time.

 Dear Dr Mark

The R/Rfree values seem very low for a 2.67Å resolution structure. Perhaps
> it was over-refined? Difficult to know without looking at the specific case
> and details of the data quality though.
>

PFA merging stats. Asymmetric unit seems to have 8 molecules @~44% bulk
solvent content. I can only speculate as to systematic problems arising in
intensity correction accompanying the MR run on account of a combination of
NCS (~26% off-origin peak in Patterson map) and a screw axis symmetry (P 1
21 1 being the space group).

The best way to resolve validation concerns is to inspect the outliers
> individually - it might be worth getting a second pair of experienced eyes
> to look at them. Perhaps there is a peptide flip or a cis-peptide that you
> missed.
> Finally, it is not usually possible to remove all validation concerns,
> especially at intermediate resolution like 2.67Å.
>

Among the clashes and the other geometry outliers, there are cis-prolines
in the final model - same as the MR model that was pulled from RCSB. As
such, I decided to leave them in. Trying to fix them in coot serves no
good, as they come undone upon uploading the resultant structure to wwPDB.
Over-fitting, as you pointed out, was also a result of the umpteen attempts
to iron these issues out with refinement/remodelling.

Dear Professor Eleanor

I use the validation report to check for obvious modelling errors but if
> you can’t find any, you have to just submit the results of your
> experiment...
>
I have sent a message to wwPDB curator requesting to admit the current
model - with outliers. Response awaited.

Dear Dr Powell

I will submit the data to PDB-REDO

Dear Professor Cooper

Many outliers have been done away with. The ones that remain seem truly
intractable. We have been trying to submit the structure in its current
state barring only the red exclamation point on the validation reports
section, even with green ticks on every other section.

Thank you all for the helpful thoughts, again. Please revert for further
information.
Yours faithfully
Nitin Kulhar

On Mon, Sep 25, 2023 at 5:44 PM Jon Cooper 
wrote:

> Hello, unless it's changed over the last couple of years, the pdb rightly
> used to let you deposit a structure with outliers, hopefully not too many,
> though ;-0
>
> I take it the stalling you reported probably comes from whoever is looking
> over your shoulder.
>
> Best wishes, Jon Cooper. jon.b.coo...@protonmail.com
>
> Sent from Proton Mail mobile
>
>
>
>  Original Message 
> On 23 Sept 2023, 02:29, Nitin Kulhar <
> 9dfccc771c91-dmarc-requ...@jiscmail.ac.uk> wrote:
>
>
> Dear all
>
> We have refined (Refmac5) a crystallographic structure with Rw/Rf values
> 0.19/0.22 (Resln 2.67). However, the deposition has stalled on account of
> the wwPDB's preliminary validation report, which indicates map/model and
> geometry issues, with each criterion containing a few instances. We tried
> to correct these by varying overall geometry restraint weights, e.g.
> decreasing overall weights from default value of 1.0, incementally
> decreasing sigmas corresponding to the planarity restraint term. This did
> not resolve the issues.
>
> In another approach, real space refinement by hand (against 2fo-fc) in
> coot brought the geometry parameters within acceptable limits in addition
> to improved apparent agreement with electron density (2fo-fc, sigma=1), but
> uploading the resultant coordinates seems to undo the changes made in coot,
> as indicated by reappearance of same outliers in the subsequent validation
> report.
>
> I request your kind suggestions in this regard. Please also revert for any
> further information.
>
> Thanks
> Nitin Kulhar
> PhD student
> c/o Dr Rajakumara Eerappa
> Macromolecular Structural Biology Group
> Department of Biotechnology
> Indian Institute of Technology Hyderabad
> Kandi, Sangareddy
> Telangana, India 502284
>
> Disclaimer:- This footer text is to convey that this email is sent by one
> of the users of IITH. So, do not mark it as SPAM.
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
>

-- 


Disclaimer:- This footer text is to convey that this email is sent by one 
of the 

Re: [ccp4bb] Register now: Stanford Electron Microscopy-X (EM-X) Symposium October 2, 2023

[Polizzi, JoAnn]

Friendly Reminder to register for the Stanford EM-X symposium on October 2, 
2023.

Best,
JoAnn 


From: chiula...@slac.stanford.edu  on behalf of 
Polizzi, JoAnn <021050d09cb7-dmarc-requ...@listserv.slac.stanford.edu>
Sent: Friday, September 22, 2023 8:10 AM
To: 3...@ncmir.ucsd.edu <3...@ncmir.ucsd.edu>; cryoem-announce 
; chiulab-l 
Subject: Register now: Stanford Electron Microscopy-X (EM-X) Symposium October 
2, 2023


Dear Colleagues,



We are pleased to invite you to our October 2023 Stanford Electron Microscopy-X 
(EM-X) Symposium on Monday, October 2nd, 2023, 8:00-9:30 AM (PST).



Speakers:

Prof. Stephen K. Burley

Professor at Rutgers, The State University of New Jersey

Talk Title: Cryo-EM Through the Lens of the Protein Data Bank: Impact of the 
Resolution Revolution and Implications for the Future

Bio: Stephen Burley currently serves as Henry Rutgers Chair and University 
Professor, Founding Director of the Institute for Quantitative Biomedicine, and 
Director of the RCSB Protein Data Bank at Rutgers, The State University of New 
Jersey. He is also a Member of the Rutgers Cancer Institute of New Jersey, 
where he Co-Leads the Cancer Pharmacology Research Program. Burley is an expert 
in structural biology, proteomics, bioinformatics, structure/fragment-based 
drug discovery, and clinical medicine/oncology.



Prof. Dan Gianola

Professor at the University of California, Santa Barbara

Talk Title: Characterizing pathways for plastic deformation using electron 
microscopy

Bio: Daniel Gianola is Professor of Materials at UCSB, where his research group 
specializes in deformation at the micro- and nanoscale, particularly using 
in-situ nanomechanical testing techniques. He has also served as the faculty 
director of the Microscopy and Microanalysis Facility at UCSB, which is a 
central shared facility with over 300 active users. He is the recipient of the 
National Science Foundation CAREER, Department of Energy Early Career, TMS 
Early Career Faculty Fellow, and Alexander von Humboldt Postdoctoral Fellow 
awards.



We expect a large attendance, please register soon at 
https://stanford.zoom.us/webinar/register/WN_g2SgqrB2R7Ke2p7Pmjre_w and visit 
our official website at https://emx.stanford.edu/ and 
https://emx.stanford.edu/previous-speakers to see our previous speakers.





We are looking forward to seeing you there!


[cid:5c0aefea-a3da-495b-baa8-e179f37e285e]



Use REPLY-ALL to reply to list

To unsubscribe from the CHIULAB-L list, click the following link:
https://listserv.slac.stanford.edu/cgi-bin/wa?SUBED1=CHIULAB-L=1



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

Re: [ccp4bb] Intractable outliers in wwPDB validation report

[Jon Cooper]

Hello, unless it's changed over the last couple of years, the pdb rightly used 
to let you deposit a structure with outliers, hopefully not too many, though ;-0

I take it the stalling you reported probably comes from whoever is looking over 
your shoulder.

Best wishes, Jon Cooper. jon.b.coo...@protonmail.com

Sent from Proton Mail mobile

 Original Message 
On 23 Sept 2023, 02:29, Nitin Kulhar wrote:

> Dear all
>
> We have refined (Refmac5) a crystallographic structure with Rw/Rf values 
> 0.19/0.22 (Resln 2.67). However, the deposition has stalled on account of the 
> wwPDB's preliminary validation report, which indicates map/model and geometry 
> issues, with each criterion containing a few instances. We tried to correct 
> these by varying overall geometry restraint weights, e.g. decreasing overall 
> weights from default value of 1.0, incementally decreasing sigmas 
> corresponding to the planarity restraint term. This did not resolve the 
> issues.
>
> In another approach, real space refinement by hand (against 2fo-fc) in coot 
> brought the geometry parameters within acceptable limits in addition to 
> improved apparent agreement with electron density (2fo-fc, sigma=1), but 
> uploading the resultant coordinates seems to undo the changes made in coot, 
> as indicated by reappearance of same outliers in the subsequent validation 
> report.
>
> I request your kind suggestions in this regard. Please also revert for any 
> further information.
>
> Thanks
> Nitin Kulhar
> PhD student
> c/o Dr Rajakumara Eerappa
> Macromolecular Structural Biology Group
> Department of Biotechnology
> Indian Institute of Technology Hyderabad
> Kandi, Sangareddy
> Telangana, India 502284
>
> Disclaimer:- This footer text is to convey that this email is sent by one of 
> the users of IITH. So, do not mark it as SPAM.
>
> ---
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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

Re: [ccp4bb] Intractable outliers in wwPDB validation report

[Harry Powell]

Hi

I think It may be worthwhile submitting your model and data to PDB-REDO to see 
if (inter alia) you have applied any unconscious bias to your building that has 
resulted in these outliers, or over- or under-interpreted the data. It’s quick 
and easy to submit and requires remarkably little effort on your part - another 
tool to your collection.

Harry

> On 25 Sep 2023, at 07:00, Guillaume Gaullier  
> wrote:
> 
> A good example of such expected outliers can be found in structures of 
> nucleosomes: the validation report always flags almost all DNA bases as 
> geometry outliers. But we have no end of evidence that these structures are 
> correct: for instance, the many ensemble and single-molecule FRET studies of 
> nucleosomes that relied on said structures to design their labeling scheme 
> and to formulate testable hypotheses.
> 
> If your map insists on putting some residues in an uncomfortable 
> conformation, it’s possible that this is indeed what is going on in the 
> protein and not necessarily a modeling error.
> 
> Cheers,
> 
> Guillaume
> 
>> On 23 Sep 2023, at 21:50, Eleanor Dodson 
>> <176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:
>> 
>> 
>> Well - some outliers are infix able! There are various reasons - floppy 
>> residues like arg or lys 
>> In multiple positions; strain due to protein folding constraints, etc.
>> 
>> I use the validation report to check for obvious modelling errors but if you 
>> can’t find any, you have to just submit the results of your experiment... 
>> 
>> On Sat, 23 Sep 2023 at 02:29, Nitin Kulhar 
>> <9dfccc771c91-dmarc-requ...@jiscmail.ac.uk> wrote:
>> Dear all
>> 
>> We have refined (Refmac5) a crystallographic structure with Rw/Rf values 
>> 0.19/0.22 (Resln 2.67). However, the deposition has stalled on account of 
>> the wwPDB's preliminary validation report, which indicates map/model and 
>> geometry issues, with each criterion containing a few instances. We tried to 
>> correct these by varying overall geometry restraint weights, e.g. decreasing 
>> overall weights from default value of 1.0, incementally decreasing sigmas 
>> corresponding to the planarity restraint term. This did not resolve the 
>> issues.
>> 
>> In another approach, real space refinement by hand (against 2fo-fc) in coot 
>> brought the geometry parameters within acceptable limits in addition to 
>> improved apparent agreement with electron density (2fo-fc, sigma=1), but 
>> uploading the resultant coordinates seems to undo the changes made in coot, 
>> as indicated by reappearance of same outliers in the subsequent validation 
>> report.
>> 
>> I request your kind suggestions in this regard. Please also revert for any 
>> further information.
>> 
>> Thanks
>> Nitin Kulhar
>> PhD student
>> c/o Dr Rajakumara Eerappa
>> Macromolecular Structural Biology Group
>> Department of Biotechnology
>> Indian Institute of Technology Hyderabad
>> Kandi, Sangareddy
>> Telangana, India 502284
>> 
>> Disclaimer:-
>> 
>> This footer text is to convey that this email is sent by one of the users of 
>> IITH. So, do not mark it as SPAM.
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>> 
>> 
>> VARNING: Klicka inte på länkar och öppna inte bilagor om du inte känner igen 
>> avsändaren och vet att innehållet är säkert.
>> CAUTION: Do not click on links or open attachments unless you recognise the 
>> sender and know the content is safe.
> 
> 
> 
> 
> 
> 
> 
> 
> När du har kontakt med oss på Uppsala universitet med e-post så innebär det 
> att vi behandlar dina personuppgifter. För att läsa mer om hur vi gör det kan 
> du läsa här: http://www.uu.se/om-uu/dataskydd-personuppgifter/ 
> 
> E-mailing Uppsala University means that we will process your personal data. 
> For more information on how this is performed, please read here: 
> http://www.uu.se/en/about-uu/data-protection-policy
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> 



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

Re: [ccp4bb] Intractable outliers in wwPDB validation report

[Guillaume Gaullier]

A good example of such expected outliers can be found in structures of 
nucleosomes: the validation report always flags almost all DNA bases as 
geometry outliers. But we have no end of evidence that these structures are 
correct: for instance, the many ensemble and single-molecule FRET studies of 
nucleosomes that relied on said structures to design their labeling scheme and 
to formulate testable hypotheses.

If your map insists on putting some residues in an uncomfortable conformation, 
it’s possible that this is indeed what is going on in the protein and not 
necessarily a modeling error.

Cheers,

Guillaume

On 23 Sep 2023, at 21:50, Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:


Well - some outliers are infix able! There are various reasons - floppy 
residues like arg or lys
In multiple positions; strain due to protein folding constraints, etc.

I use the validation report to check for obvious modelling errors but if you 
can’t find any, you have to just submit the results of your experiment...

On Sat, 23 Sep 2023 at 02:29, Nitin Kulhar 
<9dfccc771c91-dmarc-requ...@jiscmail.ac.uk>
 wrote:
Dear all

We have refined (Refmac5) a crystallographic structure with Rw/Rf values 
0.19/0.22 (Resln 2.67). However, the deposition has stalled on account of the 
wwPDB's preliminary validation report, which indicates map/model and geometry 
issues, with each criterion containing a few instances. We tried to correct 
these by varying overall geometry restraint weights, e.g. decreasing overall 
weights from default value of 1.0, incementally decreasing sigmas corresponding 
to the planarity restraint term. This did not resolve the issues.

In another approach, real space refinement by hand (against 2fo-fc) in coot 
brought the geometry parameters within acceptable limits in addition to 
improved apparent agreement with electron density (2fo-fc, sigma=1), but 
uploading the resultant coordinates seems to undo the changes made in coot, as 
indicated by reappearance of same outliers in the subsequent validation report.

I request your kind suggestions in this regard. Please also revert for any 
further information.

Thanks
Nitin Kulhar
PhD student
c/o Dr Rajakumara Eerappa
Macromolecular Structural Biology Group
Department of Biotechnology
Indian Institute of Technology Hyderabad
Kandi, Sangareddy
Telangana, India 502284


Disclaimer:- This footer text is to convey that this email is sent by one of 
the users of IITH. So, do not mark it as SPAM.



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



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


VARNING: Klicka inte på länkar och öppna inte bilagor om du inte känner igen 
avsändaren och vet att innehållet är säkert.
CAUTION: Do not click on links or open attachments unless you recognise the 
sender and know the content is safe.








När du har kontakt med oss på Uppsala universitet med e-post så innebär det att 
vi behandlar dina personuppgifter. För att läsa mer om hur vi gör det kan du 
läsa här: http://www.uu.se/om-uu/dataskydd-personuppgifter/

E-mailing Uppsala University means that we will process your personal data. For 
more information on how this is performed, please read here: 
http://www.uu.se/en/about-uu/data-protection-policy



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