Re: Question about NMR complex
Lee, Woonghee
Dear Margaret,
To make RMSD low, you must have the sufficient number of distance restraints. Usually, I advise to obtain restraints as many as possible and sort violating restraints out because getting more restraints manually while you have fuzzy structure is difficult. To do so, you have to run AUDANA automation with NOESY data and confident restraints and/or .wlt/.blt files iteratively. If your structure is well-structured, you should see well-compact superimposed structures no matter how many violations you have before getting into serious violation removal.
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
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)
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From: margaret phillips <margaretp...@gmail.com>
Date: Wednesday, July 21, 2021 at 7:08 AM
To: "Lee, Woonghee" <WOONGH...@UCDENVER.EDU>
Subject: Re: Question about NMR complex
[External Email - Use Caution]
Hi Woonghee,
Good Morning!
I am trying to reduce the backbone RMSD of the PONDEROSA structure. I notice some residues have like 20 # of viol. So I try to look at those residues using the "view in spectrum" and "view in space" options on the PONDEROSA Analyzer. I am trying to understand if this is the correct way to bring the rmsd down or am I missing something?
Also, when I look at the 'bundle.png' in the best evaluated folder, I see that out of the four alpha helices in my structure only one alpha helix has the best superimposition of all conformers. From my understanding shouldn't all the 20 conformers superimpose well to get a better structural model? How do we improve the superimposition?
Thank you.
Best,
Margaret
On Wed, Jul 14, 2021 at 3:31 PM Lee, Woonghee <WOONGH...@ucdenver.edu> wrote:
Dear Margaret,
You can just add the pdb file as a restraint.
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
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: margaret phillips <margaretp...@gmail.com>
Date: Wednesday, July 14, 2021 at 1:30 PM
To: "Lee, Woonghee" <WOONGH...@UCDENVER.EDU>
Subject: Re: Question about NMR complex
[External Email - Use Caution]
Hi Woonghee,
I am curious if there is a way to generate distance restraint file from an x-ray structure to help with identifying long range NOEs?
Thanks,
Margaret
On Fri, Jul 9, 2021 at 9:16 AM Lee, Woonghee <WOONGH...@ucdenver.edu> wrote:
Hi Margaret,
You can just assign in "at" window in POKY or SPARKY. Save the peak list and use it. In Ponderosa Client or Poky Structure Builder, you will see "preprocessed.upl" appearing in the restraint section.
Best,
Woonghee
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From: margaret phillips <margaretp...@gmail.com>
Sent: Friday, July 9, 2021 7:05:22 AM
To: Lee, Woonghee <WOONGH...@UCDENVER.EDU>
Subject: Re: Question about NMR complex
[External Email - Use Caution]
Hi Woonghee,
Good Morning!
I have been trying to get my NOESY signals assigned by PONDEROSA. I can see that there are several good intensity signals in the 15N NOESY spectrum that have not been assigned by PONDEROSA. Is there another way to assign these peaks?
Thanks.
Best,
Margaret
On Fri, May 14, 2021 at 10:00 AM margaret phillips <margaretp...@gmail.com> wrote:
Thank you so much!
On Fri, May 14, 2021 at 9:55 AM Lee, Woonghee <WOONGH...@ucdenver.edu> wrote:
Hi Margaret,
In the BestEvaluated folder, you have files end with “.assigned.list”. You can load them using “rp”.
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
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: margaret phillips <margaretp...@gmail.com>
Date: Friday, May 14, 2021 at 7:54 AM
To: "Lee, Woonghee" <WOONGH...@UCDENVER.EDU>
Subject: Re: Question about NMR complex
Hi Woonghee,
If PONDEROSA helps in picking and assigning the 3D NOESY peaks then is there a way to open and visually inspect those assignments?
I want to open the 3D NOESY strip and see what is assigned and compare it with the assigned 2D NHSQC.
Thank you.
Best,
Margaret
On Wed, May 12, 2021 at 4:10 PM Lee, Woonghee <WOONGH...@ucdenver.edu> wrote:
Probably you mistyped your email in the Ponderosa Client or Poky Builder.
--
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
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: margaret phillips <margaretp...@gmail.com>
Date: Wednesday, May 12, 2021 at 2:08 PM
To: "Lee, Woonghee" <WOONGH...@UCDENVER.EDU>
Subject: Re: Question about NMR complex
Hi Woonghee,
Usually when I submit jobs to PONDEROSA I almost immediately get an email saying =job submitted.
Today it's been a few hours and I haven't received any email yet. Is this normal? Just wanted to make sure.
Thank you,
Margaret
On Wed, May 12, 2021 at 9:21 AM Lee, Woonghee <WOONGH...@ucdenver.edu> wrote:
No problem.
--
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
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: margaret phillips <margaretp...@gmail.com>
Date: Wednesday, May 12, 2021 at 6:32 AM
To: "Lee, Woonghee" <WOONGH...@UCDENVER.EDU>
Subject: Re: Question about NMR complex
Thanks Woonghee,
I appreciate all your help!
Best,
Margaret
On Wed, May 12, 2021, 8:29 AM Lee, Woonghee <WOONGH...@ucdenver.edu> wrote:
You can try LACS but it is statistical comparison. You have to see the strip plot for backbone and see different spectra and make sure your assignments agree on them. If you have ctHSQC, that will be helpful on determining atom types by seeing plus and negative signs. Still, they require many manual works.
From: margaret phillips <margaretp...@gmail.com>
Sent: Wednesday, May 12, 2021 6:25:10 AM
To: Lee, Woonghee <WOONGH...@UCDENVER.EDU>
Subject: Re: Question about NMR complex
Thanks Woonghee,
Yes it makes sense.
Is there a way to check the correctness of assignments I have made for the backbone resonances?
Best,
Margaret
On Wed, May 12, 2021, 8:22 AM Lee, Woonghee <WOONGH...@ucdenver.edu> wrote:
Hi Margaret,
Completeness and accuracy are two measures. Making sure you have near complete assignments for backbone and sidechain atoms and they must be correct.
Woonghee
From: margaret phillips <margaretp...@gmail.com>
Sent: Wednesday, May 12, 2021 6:19:54 AM
To: Lee, Woonghee <WOONGH...@UCDENVER.EDU>
Subject: Re: Question about NMR complex
Hi Woonghee,
Could you please elaborate on what is good chemical shift?
Also, I thought PONDEROSA helps with assigning the NOESY data? Or is there another software for assigning NOESY peaks?
Thank you
Margaret
On Wed, May 12, 2021, 8:14 AM Lee, Woonghee <WOONGH...@ucdenver.edu> wrote:
Hi Margaret,
It is simple. "Junk in junk out." You have to make sure you are providing good chemical shifts and quality noesy data. Some assigned long range NOE will be helpful, too.
Best,
Woonghee
From: margaret phillips <margaretp...@gmail.com>
Sent: Wednesday, May 12, 2021 6:10:46 AM
To: Lee, Woonghee <WOONGH...@UCDENVER.EDU>
Subject: Re: Question about NMR complex
Hi Woonghee,
I am trying to use PONDEROSA to assign the NOESY peaks and get the structure of my protein.
Attached are the images of the x-ray structure I submitted in the PONDEROSA (submitted_pdb) and the image of the "final.pdb" I get after my first run.
Why is my protein final.pdb structure so open?
Is it because I need more NOE assignments (distance restraints) ?
I did include the only disulfide bond we know of but it doesn't help much.
Thank you.
Best,
Margaret
On Wed, May 5, 2021 at 3:41 PM Lee, Woonghee <WOONGH...@ucdenver.edu> wrote:
A-ha. I got it.
If you don’t put anything, Ponderosa Server will find homologous structures from PDB and try to use the information. If you provide PDB code, it will use that. If you want to use a PDB file that you have, you can add it as a restraint. No matter what method you use, you want to use the most similar structure to your structure.
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
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: margaret phillips <margaretp...@gmail.com>
Date: Wednesday, May 5, 2021 at 1:38 PM
To: "Lee, Woonghee" <WOONGH...@UCDENVER.EDU>
Subject: Re: Question about NMR complex
Sorry Woonghee,
There is an option to use "pdb id" in PONDEROSA client (next to the PACSY option). I was referring to that. Does it make any difference if we put in a pdb id there or it doesn't matter?
On Wed, May 5, 2021 at 3:34 PM Lee, Woonghee <WOONGH...@ucdenver.edu> wrote:
Hi Margaret,
I am sorry I don’t understand your question.
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
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: margaret phillips <margaretp...@gmail.com>
Date: Wednesday, May 5, 2021 at 1:32 PM
To: "Lee, Woonghee" <WOONGH...@UCDENVER.EDU>
Subject: Re: Question about NMR complex
Hi Woonghee,
One more question. If I am using PONDEROSA for my complex NOESY spectrum assignment and structure calculations, is it better to use the "protein-complex.pdb" file or I can use the "apo protein.pdb"? There is no structure for the complex I am working with.
Thank you,
Best,
Margaret
On Tue, May 4, 2021 at 5:54 PM Lee, Woonghee <WOONGH...@ucdenver.edu> wrote:
Dear Margaret,
Making a complex structure is an extra step after you make a holo-like structure. That will involve in operating manual Xplor-NIH or using HADDOCK. You should have your peptide structure optimized, too. But, you can think about it later though.
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
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: margaret phillips <margaretp...@gmail.com>
Date: Tuesday, May 4, 2021 at 3:52 PM
To: "Lee, Woonghee" <WOONGH...@UCDENVER.EDU>
Subject: Question about NMR complex
Hi Woonghee,
How do we build in a peptide structure into a protein-peptide complex? The NOESY and other 3D experiments I have are for the complex but when I use PONDEROSA for NOESY peak picking and structure calculations I only see the protein backbone in the best evaluated folder.
Do we need to record separate experiments for the peptide?
Best,
Margaret
Lee, Woonghee
Dear Margaret,
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861749/
See the figure 14. You can assist AUDANA to assign NOE cross peaks correctly if it is ambiguous and has multiple candidates by promoting it and demoting it. You can open the tool by right-clicking the main grid of Poky Analyzer (formerly Ponderosa Analyzer). I mean “confident restraints” are the significant long-range restraints that you are pretty sure they are correct. If you add a few important restraints as additional inputs (.upl) when running automated NOE assignment (AUDANA), they can work as decoys and assist better NOE assignment.
Hi Woonghee,
Thank you for your reply.
I am not sure I understand what the ".wlt/.blt " means? Where/How do I obtain these files?
Also, What is "confident restraint"?
Thank you.
Best,
Margaret
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