poky analyzer questions

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Jun Yang

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Aug 17, 2021, 4:01:41 PM8/17/21
to NMR POKY/SPARKY USER GROUP
I was overwhelmed by the content of first round of calculation result. It really takes me a while to fully understand. Unfortunately it is difficult to search for the analyzer tutorial section from the 6~7-hour long workshop. The individual ponderosa videos are not very helpful. I don't think a new user can get much from following just the mouse clicks without verbal explanations.

After many try and error, what I understand so far is that we need to validate the distance constraints and angle constraints. Right now I'm focusing on distance constrains and here are the questions:

0. During the poky builder, I didn't specify a template pdb file, so the 1st round of calculation was based on a random coil structure, right?
1. What special about restraints in "Strictly used restraints" window?I checked them in the validator and some of them have no violation;
2. In the "Poky Analyzer Connector" window, what type of spectra are for "2D CC"? What do the buttons of "Move(NNOE)", "Move(CNOE)" do?
3. Because only one 13C-noesy dataset is allowed, I have to use the one with aliased Calpha. Therefore, when I click a list item that has the Ca shift outside the dataset, it generates the error about invalid shift. In poky, it will automatically subtract one spectral width and locate the right position, can it be fixed so that it will do the same in analyzer?
4. For constraints that I validate, do I need to assign them in the corresponding spectra? If so, how to do that?
5. In several cases, I saw the constraints are degenerated with other validate assignment, eg, sequential Ha-H with intra Ha-H. Do I validate them?

Thanks.

Jun

Lee, Woonghee

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Aug 17, 2021, 4:32:29 PM8/17/21
to Jun Yang, NMR POKY/SPARKY USER GROUP

Dear Jun,

 

I understand your frustration. But that is what it is. NMR structure calculation is not a simple thing. Some might misjudge structure is done thing after finishing resonance assignments. Apparently NOT! NMRFAM workshop materials are really well-organized. Usually, a scientist spends a whole week and performs hands-on with the many staff scientists’ assistance. If you finish everything, you will be at least capable of determining a simple and small protein like ubiquitin. But every proteins behave differently and often one encounters a wall when analyzing their own. That is natural. Because no one is responsible for your protein, it will be you who struggles to figure out the path. However, it is nearly impossible if you don’t have anyone with the sufficient experience on that already around you. But I answer your questions and hope they are helpful.

 

  1. Yes and no. Not a random coil but a stretched thread structure.
  2. They have very high score from the scratch and they might not be kicked out of the restraint set by modest violations. That means you may want to double-check they are correct from your NOESY data and make sense from calculated 3D structure.
  3. 2D CC is ssNMR not yours. Move buttons act the same as you double-click on the labels. If you double-click and it is okay, you don’t need to use those buttons.
  4. No. That’s the reason why I don’t like to collect the data folded. You probably want to make your data unfolded using nmrpipe like Gabriel suggested a few weeks ago.
  5. It depends. If you will include validated constraints in the next round and also use noesy peak lists as inputs, you don’t have to. If you will use only peak lists not constraints, you probably want to assign. You can just do that in the “at” window.
  6. It is up to do. Sequential & Intra assignments are not really critical to get the structure folded in general. Focus more on long ranges and typical secondary structure forming restraints.

 

Best,

Woonghee

 

 

 

 

--

Woonghee Lee, I.E.I.P., M.S., Ph.D.

 

Assistant Professor

Department of Chemistry

University of Colorado Denver

1151 Arapahoe St. (Science Bldg.) Rm 4128A

Denver, CO 80217-3364, USA

Office: +1-303-315-7672

woongh...@ucdenver.edu

 

https://poky.clas.ucdenver.edu

https://poky.clas.ucdenver.edu/wlee-group

https://clas.ucdenver.edu/chemistry/woonghee-lee

 

Shipping/Mailing Address:

Woonghee Lee

1201 5th St. UCD CHEM-194

P.O. Box 173364 (USPS)

Denver, CO 80204, USA

 

From: <nmr-s...@googlegroups.com> on behalf of Jun Yang <yangj...@gmail.com>
Date: Tuesday, August 17, 2021 at 2:01 PM
To: NMR POKY/SPARKY USER GROUP <nmr-s...@googlegroups.com>
Subject: [NMR POKY/SPARKY] poky analyzer questions

 

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Jun Yang

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Aug 17, 2021, 5:02:18 PM8/17/21
to NMR POKY/SPARKY USER GROUP
Hi Woonghee,

Thanks for your information and encouragement. The distance/angle constraint validator are from file "noe.tbl" and "aco.tbl", right? I'll just convert it to a list file and read into the spectrum and check. Do you get a change to check the "ot" issue?

Bests,

Jun

Jun Yang

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Aug 17, 2021, 5:28:58 PM8/17/21
to NMR POKY/SPARKY USER GROUP
I found there are some differences between the noe.tbl and the table:
1. all carbon-attached protons ending with a star:
2. all proton ending with "3" in the table becomes "1" in the noe.tbl;
3. all "H" become "HN" in noe.tbl;
3. constraints involving oxygen don't agree in the file and in the table;

Is that all the differences I should expect?

Thanks

Jun

Lee, Woonghee

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Aug 17, 2021, 5:57:48 PM8/17/21
to Jun Yang, NMR POKY/SPARKY USER GROUP

Hi Jun,

 

There is a nomenclature discrepancy between XPLOR-NIH and BMRB. You will see that noe.tbl and final.upl are similar but you will also see the nomenclature difference. * and 1/2 <-> 2/3 are tricks to make XPLOR-NIH compatible with BMRB nomenclature. Basically, Ponderosa Server program automatically converts them and you don’t have to worry about it.

 

You will also see .assigned.list files in the BestEvaluated directory. You can use “rp” to read the file in.

 

“ot” issue has been resolved in the version 08/17/2021.

 

Best,

Woonghee   

 

--

Woonghee Lee, I.E.I.P., M.S., Ph.D.

 

Assistant Professor

Department of Chemistry

University of Colorado Denver

1151 Arapahoe St. (Science Bldg.) Rm 4128A

Denver, CO 80217-3364, USA

Office: +1-303-315-7672

woongh...@ucdenver.edu

 

https://poky.clas.ucdenver.edu

https://poky.clas.ucdenver.edu/wlee-group

https://clas.ucdenver.edu/chemistry/woonghee-lee

 

Shipping/Mailing Address:

Woonghee Lee

1201 5th St. UCD CHEM-194

P.O. Box 173364 (USPS)

Denver, CO 80204, USA

 

From: <nmr-s...@googlegroups.com> on behalf of Jun Yang <yangj...@gmail.com>
Date: Tuesday, August 17, 2021 at 3:29 PM
To: NMR POKY/SPARKY USER GROUP <nmr-s...@googlegroups.com>
Subject: Re: [NMR POKY/SPARKY] poky analyzer questions

 

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I found there are some differences between the noe.tbl and the table:

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Jun Yang

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Aug 17, 2021, 6:26:38 PM8/17/21
to NMR POKY/SPARKY USER GROUP
Thanks.   Jun

Jun Yang

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Aug 18, 2021, 12:25:20 PM8/18/21
to NMR POKY/SPARKY USER GROUP
Hi Woonghee,

What's the difference between final.pdb and finalx.pdb?

Also it seems to me that the pymol command "f1" should remove the space between plus and the residue index to work, that is:
intra_fit i. 136-173+ 196-200+ 234-240+ 263-318
align final_0002 & i. 136-173+ 196-200+ 234-240+ 263-318, final_0001, cutoff=0
should be
intra_fit i. 136-173+196-200+234-240+263-318
align final_0002 & i. 136-173+196-200+234-240+263-318, final_0001, cutoff=0
Is that so?

Thanks.

Jun

Lee, Woonghee

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Aug 18, 2021, 12:33:23 PM8/18/21
to Jun Yang, NMR POKY/SPARKY USER GROUP

Hi Jun,

 

Nomenclature of finalx.pdb is 1/2 system and that of final.pdb is 2/3 system.

That f1 thing, the space will be removed. You don’t have to update Poky because it is Ponderosa Server. There are running jobs now and that will be corrected if nothing is running.

 

Best,

Woonghee

--

Woonghee Lee, I.E.I.P., M.S., Ph.D.

 

Assistant Professor

Department of Chemistry

University of Colorado Denver

1151 Arapahoe St. (Science Bldg.) Rm 4128A

Denver, CO 80217-3364, USA

Office: +1-303-315-7672

woongh...@ucdenver.edu

 

https://poky.clas.ucdenver.edu

https://poky.clas.ucdenver.edu/wlee-group

https://clas.ucdenver.edu/chemistry/woonghee-lee

 

Shipping/Mailing Address:

Woonghee Lee

1201 5th St. UCD CHEM-194

P.O. Box 173364 (USPS)

Denver, CO 80204, USA

 

 

From: <nmr-s...@googlegroups.com> on behalf of Jun Yang <yangj...@gmail.com>
Date: Wednesday, August 18, 2021 at 10:25 AM
To: NMR POKY/SPARKY USER GROUP <nmr-s...@googlegroups.com>
Subject: Re: [NMR POKY/SPARKY] poky analyzer questions

 

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Hi Woonghee,

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Jun Yang

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Aug 20, 2021, 7:19:35 PM8/20/21
to NMR POKY/SPARKY USER GROUP
After comparing the noe.tbl and n/c-noesy.assigned.list, I notice that at least there are constraints in noe.tbl that are not found in the list file. Is that normal? Also why are there quite a bit diagonal peaks assigned in the list file, and also some noe constraints among methylene protons?

Thanks.

Jun

Lee, Woonghee

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Aug 20, 2021, 7:26:34 PM8/20/21
to Jun Yang, NMR POKY/SPARKY USER GROUP

Hi Jun,

 

For the restraint residue number I to j, you should check residue number j to I as well because numbers in the restraints are ordered.

 

Also why are there quite a bit diagonal peaks assigned in the list file?

 

Diagonal peaks can be assigned with less doubts whether they were used to generate restraints or not for the calculation.

and also some noe constraints among methylene protons?

 

I don’t see a problem for that. What do you mean?

 

Best,

Woonghee

 

--

Woonghee Lee, I.E.I.P., M.S., Ph.D.

 

Assistant Professor

Department of Chemistry

University of Colorado Denver

1151 Arapahoe St. (Science Bldg.) Rm 4128A

Denver, CO 80217-3364, USA

Office: +1-303-315-7672

woongh...@ucdenver.edu

 

https://poky.clas.ucdenver.edu

https://poky.clas.ucdenver.edu/wlee-group

https://clas.ucdenver.edu/chemistry/woonghee-lee

 

Shipping/Mailing Address:

Woonghee Lee

1201 5th St. UCD CHEM-194

P.O. Box 173364 (USPS)

Denver, CO 80204, USA

 

From: <nmr-s...@googlegroups.com> on behalf of Jun Yang <yangj...@gmail.com>
Date: Friday, August 20, 2021 at 5:19 PM
To: NMR POKY/SPARKY USER GROUP <nmr-s...@googlegroups.com>
Subject: Re: [NMR POKY/SPARKY] poky analyzer questions

 

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After comparing the noe.tbl and n/c-noesy.assigned.list, I notice that at least there are constraints in noe.tbl that are not found in the list file. Is that normal? Also why are there quite a bit diagonal peaks assigned in the list file, and also some noe constraints among methylene protons?

 

Thanks.

 

Jun

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Jun Yang

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Aug 26, 2021, 9:35:36 AM8/26/21
to NMR POKY/SPARKY USER GROUP
After examining the noesy list files in the ponderosa results, I have some questions:

1. I found there are a lot of peak labels having the wrong heteroatom assignments, such as following. Are these some bugs in the server program?
in n-noesy list, where these Ns should be NE2 or ND2 or NE
   Q279N-Q279HE22-Q279HE22     112.778      6.861      6.861          8229712
   Q279N-Q279HE21-Q279HE21     112.778      7.736      7.736          8229712
       R281N-R281HE-R281HE     115.188      7.465      7.465          8229712
   N273N-N273HD22-N273HD22     113.778      6.880      6.880          8229712
   N273N-N273HD21-N273HD21     113.778      7.546      7.546          8229712

in c-noesy list, where these CA/B should be CD
      R281CA-R281HD-R281HD      41.995      3.158      3.158          8158815
      R282CA-R282HD-R282HD      43.500      3.161      3.161          8158815
    R230CA-R230HD2-R230HD2      42.729      3.163      3.163          8158815
      R220CB-R220HD-R220HD      43.661      3.046      3.046          8158815

2. The diagonal peaks intensities are all the same. Are they designed to be like that? That is, if I need to refresh the peak heights, should I need to avoid changing them?

3. The nomenclature of the assignment seems to drop the index value if they are degenerated, eg, if Hx2 and Hx3 are not distinguishable (either because only one proton shift is available or the peak location can not be decisively assigned even two proton shifts are provided), it'll be labeled as Hx. This applies to methyl protons, ie, A-HB# => A-HB. Is that right?

Thanks.

Jun

Lee, Woonghee

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Aug 26, 2021, 9:46:16 AM8/26/21
to Jun Yang, NMR POKY/SPARKY USER GROUP

Jun,

 

Send me your job ID privately. That will be helpful for me to look over despite it may take a while.

  1. They look fish. I think they haven’t contributed when making any restraints but looks not right. Peak list generation is almost at the end of the processes, and it uses distance restraints to simulate and match NOE cross peaks. So, Something happened when making heavy atom dimension which is not related to proton atoms used in restraints. However, I will take a look on it.
  2. When there’s a peak and peak positions are the same, it will be the position and intensity while assignments are different. That’s the reason why you see same heights.
  3. Yes, it is right. At the server side, it will add * to make A-HB*. But you will see still HB (no star) in the Analyzer program and .upl files. You can see the difference in noe.tbl and noe.tbl.upl files.

 

Best,

Woonghee

 

--

Woonghee Lee, I.E.I.P., M.S., Ph.D.

 

Assistant Professor

Department of Chemistry

University of Colorado Denver

1151 Arapahoe St. (Science Bldg.) Rm 4128A

Denver, CO 80217-3364, USA

Office: +1-303-315-7672

woongh...@ucdenver.edu

 

https://poky.clas.ucdenver.edu

https://poky.clas.ucdenver.edu/wlee-group

https://clas.ucdenver.edu/chemistry/woonghee-lee

 

Shipping/Mailing Address:

Woonghee Lee

1201 5th St. UCD CHEM-194

P.O. Box 173364 (USPS)

Denver, CO 80204, USA

 

 

From: <nmr-s...@googlegroups.com> on behalf of Jun Yang <yangj...@gmail.com>
Date: Thursday, August 26, 2021 at 7:35 AM
To: NMR POKY/SPARKY USER GROUP <nmr-s...@googlegroups.com>
Subject: Re: [NMR POKY/SPARKY] poky analyzer questions

 

[External Email - Use Caution]After examining the noesy list files in the ponderosa results, I have some questions:

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Lee, Woonghee

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Aug 26, 2021, 5:57:00 PM8/26/21
to Jun Yang, NMR POKY/SPARKY USER GROUP

Dear Jun,

 

The issue #1 must be fixed by now. You don’t have to update Poky. It is the server-side.

 

Best,

Woonghee

 

--

Woonghee Lee, I.E.I.P., M.S., Ph.D.

 

Assistant Professor

Department of Chemistry

University of Colorado Denver

1151 Arapahoe St. (Science Bldg.) Rm 4128A

Denver, CO 80217-3364, USA

Office: +1-303-315-7672

woongh...@ucdenver.edu

 

https://poky.clas.ucdenver.edu

https://poky.clas.ucdenver.edu/wlee-group

https://clas.ucdenver.edu/chemistry/woonghee-lee

 

Shipping/Mailing Address:

Woonghee Lee

1201 5th St. UCD CHEM-194

P.O. Box 173364 (USPS)

Denver, CO 80204, USA

 

Jun Yang

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Aug 27, 2021, 7:40:11 AM8/27/21
to Lee, Woonghee, NMR POKY/SPARKY USER GROUP
Got it. Thanks!

Stefano Luciano Ciurli

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Aug 27, 2021, 12:23:56 PM8/27/21
to Lee, Woonghee, NMR POKY/SPARKY USER GROUP
Hi Woonghee,

I am trying to import a BMRB assignment onto a, HSQC spectrum. I followed the tutorial here:


However, when I reach the point of giving the ‘ta’ command, I see this:


Do you know why?

I am using NMRFAM Sparky 3.190.

Thanks!
Stefano

Lee, Woonghee

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Aug 27, 2021, 12:36:20 PM8/27/21
to Stefano Luciano Ciurli, NMR POKY/SPARKY USER GROUP
Hi Stefano,

BMRB has moved to new place and the URL implemented in older versions might not work. You can find HSQC peak list from BMRB entry web page and read the file by "rp".

Best,
Woonghee
From: Stefano Luciano Ciurli <stefano...@unibo.it>
Sent: Friday, August 27, 2021 10:23:37 AM
To: Lee, Woonghee <WOONGH...@UCDENVER.EDU>; NMR POKY/SPARKY USER GROUP <nmr-s...@googlegroups.com>
Subject: Transfer assignment
 
[External Email - Use Caution] Hi Woonghee,

Stefano Luciano Ciurli

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Aug 27, 2021, 1:45:41 PM8/27/21
to Lee, Woonghee, NMR POKY/SPARKY USER GROUP
Thank you Woonghee!

Sent from my iPhone

On 27 Aug 2021, at 18:36, Lee, Woonghee <WOONGH...@ucdenver.edu> wrote:


<PastedGraphic-6.png>

Stefano Luciano Ciurli

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Aug 28, 2021, 2:51:05 AM8/28/21
to Lee, Woonghee, NMR POKY/SPARKY USER GROUP
Hi Woonghee,

I tried to read in the peaks from a list file that was made from an NMRstar file, with the command ‘rp', but I see this message>


Can you please give me the full sequence of commands to go from an assignment file downloaded manually from the BMRB (*str) to having peaks in the HSQC?

Thanks!
Stefano

<PastedGraphic-6.png>

Do you know why?

I am using NMRFAM Sparky 3.190.

Thanks!
Stefano
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Lee, Woonghee

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Aug 28, 2021, 1:36:39 PM8/28/21
to Stefano Luciano Ciurli, NMR POKY/SPARKY USER GROUP

Hi Stefano,

 

If you go your entry page, you see this at the bottom.

 

 

You download Backbone or all simulated shifts (including side chain amide groups) and read the file in using “rp”. You may need to turn the auto dimension detection on.

 

Best,

Woonghee

 

--

Woonghee Lee, I.E.I.P., M.S., Ph.D.

 

Assistant Professor

Department of Chemistry

University of Colorado Denver

1151 Arapahoe St. (Science Bldg.) Rm 4128A

Denver, CO 80217-3364, USA

Office: +1-303-315-7672

woongh...@ucdenver.edu

 

https://poky.clas.ucdenver.edu

https://poky.clas.ucdenver.edu/wlee-group

https://clas.ucdenver.edu/chemistry/woonghee-lee

 

Shipping/Mailing Address:

Woonghee Lee

1201 5th St. UCD CHEM-194

P.O. Box 173364 (USPS)

Denver, CO 80204, USA

 

 

Gabriel Cornilescu

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Aug 28, 2021, 2:47:07 PM8/28/21
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Very useful feature Woonghee, I used it a few times in the past!

I would say that you definitely need to turn the auto dimension detection on. Wonghee, wouldn't it be better to maybe have it on by default? Various processing schemes may confuse the dimension order more often than not and the rp will not complete properly with no error message as to what's wrong...

Lee, Woonghee

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Aug 28, 2021, 3:00:02 PM8/28/21
to Gabriel Cornilescu, nmr-s...@googlegroups.com
Hi Gabriel,

That is an option to consider definitely. Only problem is the automation not always works as you know well. But, yes it can be a default.

Woonghee
From: nmr-s...@googlegroups.com <nmr-s...@googlegroups.com> on behalf of Gabriel Cornilescu <gcorn...@gmail.com>
Sent: Saturday, August 28, 2021 12:47:02 PM
To: nmr-s...@googlegroups.com <nmr-s...@googlegroups.com>

Stefano Luciano Ciurli

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Aug 30, 2021, 5:02:18 AM8/30/21
to Lee, Woonghee, NMR POKY/SPARKY USER GROUP
Thank you Woonghee, it worked.
Stefano

> On 28 Aug 2021, at 19:36, Lee, Woonghee <WOONGH...@UCDENVER.EDU> wrote:
>
> <image001.png>

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