Re: [ccp4bb] MR problem in muti-domain structure

[Jon Cooper]

Hello, just to double check, I assume that you input the correctly oriented and 
positioned structure of each domain from successive runs as a 'fixed' model? 
Cheers, Jon.C.

Sent from ProtonMail mobile

 Original Message 
On 11 May 2021, 02:53, Lande wrote:

> Hi everyone, I started my work on data processing in protein crystallography 
> about half year ago and have done a fair number of “easy” structures. 
> Recently, I processed a tutorial structure containing 3 different domains and 
> two chains of them (in particular, it’s “ mth685, high resolution data” from 
> ccp4 legacy tutorial), but it was failed during MR process.
>
> According to this tutorial, I did MR with MOLREP by adding each domain in 
> order. The first and second added domains, which are domain 1 from chain A 
> and domain 2 from chain B, are correct; however, when I tried to add the next 
> domain, R work/R free did not drop and clearly MOLREP did not find the 
> correct solution according to the density map. I also tried Phaser but the 
> result was the same.
>
> screen shot for domain 2 in chain A: https://ibb.co/YB2JnRR
>
> https://ibb.co/YB2JnRR";> src="https://i.ibb.co/0yq14DD/screenshot-on-wrong-domain.png"; 
> alt="screenshot-on-wrong-domain" border="0"> href='https://imgbb.com/'>pio pio 3 menu
>
> In addition, I found an article on that data 
> (https://journals.iucr.org/d/issues/2008/01/00/ba5117/index.html) but I 
> failed to get the same result expect the first two domains.
>
> I wonder if anyone has done this tutorial before or has faced similar 
> problems in his own work. I have no idea on how to deal with it. Any 
> suggestions or comments will be helpful. Thanks in ahead.
>
> Link for that tutorial: 
> ftp://ftp.ccp4.ac.uk/tutorials/ccp4_phasing/7_domains/mth685.pdf
>
> ---
>
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Re: [ccp4bb] MR problem in muti-domain structure

[Eleanor Dodson]

Where did you find the tutorial files??
Eleanor

On Tue, 11 May 2021 at 10:24, Jon Cooper <
488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hello, just to double check, I assume that you input the correctly
> oriented and positioned structure of each domain from successive runs as a
> 'fixed' model? Cheers, Jon.C.
>
> Sent from ProtonMail mobile
>
>
>
>  Original Message 
> On 11 May 2021, 02:53, Lande < fu...@northeastern.edu> wrote:
>
>
> Hi everyone, I started my work on data processing in protein
> crystallography about half year ago and have done a fair number of “easy”
> structures. Recently, I processed a tutorial structure containing 3
> different domains and two chains of them (in particular, it’s “ mth685,
> high resolution data” from ccp4 legacy tutorial), but it was failed during
> MR process.
>
> According to this tutorial, I did MR with MOLREP by adding each domain in
> order. The first and second added domains, which are domain 1 from chain A
> and domain 2 from chain B, are correct; however, when I tried to add the
> next domain, R work/R free did not drop and clearly MOLREP did not find the
> correct solution according to the density map. I also tried Phaser but the
> result was the same.
>
> screen shot for domain 2 in chain A: https://ibb.co/YB2JnRR
>
> https://ibb.co/YB2JnRR";>https://i.ibb.co/0yq14DD/screenshot-on-wrong-domain.png";
> alt="screenshot-on-wrong-domain" border="0"> href='https://imgbb.com/'>pio pio 3 menu
>
> In addition, I found an article on that data (
> https://journals.iucr.org/d/issues/2008/01/00/ba5117/index.html) but I
> failed to get the same result expect the first two domains.
>
> I wonder if anyone has done this tutorial before or has faced similar
> problems in his own work. I have no idea on how to deal with it. Any
> suggestions or comments will be helpful. Thanks in ahead.
>
> Link for that tutorial:
> ftp://ftp.ccp4.ac.uk/tutorials/ccp4_phasing/7_domains/mth685.pdf
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>
> --
>
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>
>



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Re: [ccp4bb] MR problem in muti-domain structure

[Randy John Read]

Hi,

I had a hard time tracking down the data in the legacy CCP4 site but eventually 
found it!  Then I tried solving the MR tutorial case with Phaser.

Instead of using the tutorial domain 1, 2 and 3 files, I prepared them from the 
deposited PDB file using a sequence alignment followed by using sculptor to 
trim off non-conserved ends of side chains and any missing loops.  Then I did a 
default run of Phaser, searching for 2 copies of each domain. Phaser 
automatically set the search order to domain 1, then domain 2 then domain 3 
(they have similar sequence identities and that sorts them from biggest to 
smallest).  It was clear from the statistics (increase in log-likelihood gain 
or LLG and TFZ score) that both copies of domain 1 were correct, the first copy 
of domain 2, and both copies of domain 3, even though an incorrect placement of 
domain 2 occurred before looking for domain 3.  Unfortunately, the second copy 
of domain 3 was rejected because of packing problems with the badly-placed 
domain 2 copy.

So I went back to the partial solution with 2 copies of domain 1 and one of 
domain 2, then added both copies of domain 3 followed by the second copy of 
domain 2.  This all worked well, giving clear solutions.  No intermediate 
refinement necessary, though if it hadn’t worked it would have been possible to 
refine the intermediate solution and grab the improved models for any missing 
domains.  Fifty cycles of jelly-body refinement of the final MR solution in 
Refmac gave an R-free of 43.7%.

Note that in these jobs Phaser struggled with the second copy of domain 1, only 
pulling it out in the refinement step, probably because the B-factor had to be 
increased substantially to agree with the data.  Another search order might 
have been more efficient (maybe 1, 2, 3, 1 2, 3), but this worked.  Note that 
you can over-ride the automatic choice of search order, but Phaser will by 
default choose its own search order (which is usually but not always better).

Hope that helps on the Phaser front!

Best wishes

Randy Read

> On 11 May 2021, at 02:53, Lande  wrote:
> 
> Hi everyone, I started my work on data processing in protein crystallography 
> about half year ago and have done a fair number of “easy” structures. 
> Recently, I processed a tutorial structure containing 3 different domains and 
> two chains of them (in particular, it’s “ mth685, high resolution data” from 
> ccp4 legacy tutorial), but it was failed during MR process.
> 
> According to this tutorial, I did MR with MOLREP by adding each domain in 
> order. The first and second added domains, which are domain 1 from chain A 
> and domain 2 from chain B, are correct; however, when I tried to add the next 
> domain, R work/R free did not drop and clearly MOLREP did not find the 
> correct solution according to the density map. I also tried Phaser but the 
> result was the same.
> 
> screen shot for domain 2 in chain A: https://ibb.co/YB2JnRR
> 
> https://ibb.co/YB2JnRR";> src="https://i.ibb.co/0yq14DD/screenshot-on-wrong-domain.png"; 
> alt="screenshot-on-wrong-domain" border="0"> href='https://imgbb.com/'>pio pio 3 menu
> 
> In addition, I found an article on that data 
> (https://journals.iucr.org/d/issues/2008/01/00/ba5117/index.html) but I 
> failed to get the same result expect the first two domains.
> 
> I wonder if anyone has done this tutorial before or has faced similar 
> problems in his own work. I have no idea on how to deal with it. Any 
> suggestions or comments will be helpful. Thanks in ahead.
> 
> Link for that tutorial: 
> ftp://ftp.ccp4.ac.uk/tutorials/ccp4_phasing/7_domains/mth685.pdf
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
> 

-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: +44 1223 336500
The Keith Peters Building   Fax: +44 1223 336827
Hills Road   E-mail: 
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.  
www-structmed.cimr.cam.ac.uk




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Re: [ccp4bb] MR problem in muti-domain structure

[Randy John Read]

Brief followup:

1. Typo: Phaser struggled with the second copy of domain *2*, not domain 1.

2. I thought the single job searching for 1+1+2+3+3+2 would work, but it failed 
at the last step.  The easiest way to solve this with Phaser might be to do one 
job with 1+1+2+3+3, and then do a second job adding the second copy of domain 
2.  The reason for this, I think, is that Phaser refines all of the placed 
models simultaneously, including their relative B factors and estimated RMS 
errors, at the end of the whole job, not after placing every model.  After each 
intermediate placement, Phaser only refines the parameters of the last 
component placed and not the estimated RMS error.  The improvement the full 
refinement gives in the 5-domain solution is just enough that the second copy 
of domain 2 can finally be found.

3. If you want to find this and some other tutorials, go to 
http://legacy.ccp4.ac.uk/tutorials/targets/standard/, expand the section on 
“Basic phasing tutorials” under “ccp4i tutorials”, and download phasing.tgz, 
which contains what you need to run 9 tutorials in this section (of which this 
is #7).

Best wishes,

Randy Read

> On 11 May 2021, at 14:57, Randy John Read  wrote:
> 
> Hi,
> 
> I had a hard time tracking down the data in the legacy CCP4 site but 
> eventually found it!  Then I tried solving the MR tutorial case with Phaser.
> 
> Instead of using the tutorial domain 1, 2 and 3 files, I prepared them from 
> the deposited PDB file using a sequence alignment followed by using sculptor 
> to trim off non-conserved ends of side chains and any missing loops.  Then I 
> did a default run of Phaser, searching for 2 copies of each domain. Phaser 
> automatically set the search order to domain 1, then domain 2 then domain 3 
> (they have similar sequence identities and that sorts them from biggest to 
> smallest).  It was clear from the statistics (increase in log-likelihood gain 
> or LLG and TFZ score) that both copies of domain 1 were correct, the first 
> copy of domain 2, and both copies of domain 3, even though an incorrect 
> placement of domain 2 occurred before looking for domain 3.  Unfortunately, 
> the second copy of domain 3 was rejected because of packing problems with the 
> badly-placed domain 2 copy.
> 
> So I went back to the partial solution with 2 copies of domain 1 and one of 
> domain 2, then added both copies of domain 3 followed by the second copy of 
> domain 2.  This all worked well, giving clear solutions.  No intermediate 
> refinement necessary, though if it hadn’t worked it would have been possible 
> to refine the intermediate solution and grab the improved models for any 
> missing domains.  Fifty cycles of jelly-body refinement of the final MR 
> solution in Refmac gave an R-free of 43.7%.
> 
> Note that in these jobs Phaser struggled with the second copy of domain 1, 
> only pulling it out in the refinement step, probably because the B-factor had 
> to be increased substantially to agree with the data.  Another search order 
> might have been more efficient (maybe 1, 2, 3, 1 2, 3), but this worked.  
> Note that you can over-ride the automatic choice of search order, but Phaser 
> will by default choose its own search order (which is usually but not always 
> better).
> 
> Hope that helps on the Phaser front!
> 
> Best wishes
> 
> Randy Read
> 
>> On 11 May 2021, at 02:53, Lande  wrote:
>> 
>> Hi everyone, I started my work on data processing in protein crystallography 
>> about half year ago and have done a fair number of “easy” structures. 
>> Recently, I processed a tutorial structure containing 3 different domains 
>> and two chains of them (in particular, it’s “ mth685, high resolution data” 
>> from ccp4 legacy tutorial), but it was failed during MR process.
>> 
>> According to this tutorial, I did MR with MOLREP by adding each domain in 
>> order. The first and second added domains, which are domain 1 from chain A 
>> and domain 2 from chain B, are correct; however, when I tried to add the 
>> next domain, R work/R free did not drop and clearly MOLREP did not find the 
>> correct solution according to the density map. I also tried Phaser but the 
>> result was the same.
>> 
>> screen shot for domain 2 in chain A: https://ibb.co/YB2JnRR
>> 
>> https://ibb.co/YB2JnRR";>> src="https://i.ibb.co/0yq14DD/screenshot-on-wrong-domain.png"; 
>> alt="screenshot-on-wrong-domain" border="0">> href='https://imgbb.com/'>pio pio 3 menu
>> 
>> In addition, I found an article on that data 
>> (https://journals.iucr.org/d/issues/2008/01/00/ba5117/index.html) but I 
>> failed to get the same result expect the first two domains.
>> 
>> I wonder if anyone has done this tutorial before or has faced similar 
>> problems in his own work. I have no idea on how to deal with it. Any 
>> suggestions or comments will be helpful. Thanks in ahead.
>> 
>> Link for that tutorial: 
>> ftp://ftp.ccp4.ac.uk/tutorials/ccp4_phasing/7_domains/mth685.pdf
>> 
>> 
>> To unsubscribe from