0. An example set for demonstration

Small ubiquitin data set for running PONDEROSA-C/S is available from our [download] web page. Backbone and side chain assignments, and peak lists are all prepared by automation in the NMRFAM-SPARKY program and the I-PINE web server (two-letter-code "ae" in the NMRFAM-SPARKY).

This example section is based on the assumption that you are running linux. Not really different from the other operating system if you are not a server administrator.

1. For Users

If you have on the order of 90% of the chemical shifts assigned, you may want to obtain a 3D structure as soon as possible. Maybe you have 3D-NOESY spectra collected and processed even they are not yet picked for NOE peaks. 

What you need to do first is to run Ponderosa Client.

$ PonderosaClient [enter] or double click Ponderosa Client icon.

Set a working directory. Create a new one.

Fill in your project information. The E-mail address is essential for receiving results.

Set up inputs. URL must be the server IP of yours or localhost (with SSH tunneling) if you are NMRFAM user. You can stick with CYANA automation if your NOESY looks clean, or set it PONDEROSA refinement.

Click Analyze NOESY button and wait for 10-30 min for the initial peak identification and validation. After that, click Calculate strc.

If you see the message that you are accepted, just close the Ponderosa Client and wait for emails from Ponderosa Server. If you see the message that you are denied, contact server administrator or NMRFAM.

Soon, you will receive a notification of receipt of the job from the Ponderosa Server.

Wait for a few minutes to hours depending on your settings until you receive another notification from Ponderosa Server.

Download zip file from the URL in the email, and unzip in separate folder.

Run Ponderosa Analyzer.

$ PonderosaAnalyzer [enter] or click the icon

Set the separate folder as a working directory in the Ponderosa Analyzer. It will take a couple of minute to make a full backup of outputs in case. Only Best Evaluated will be seen at first as it is default in Calculation stage box. Try to change to All and find the best one by using sort drop down menu.

If you think Best Evaluated is the best, select that row and click Show Validation Tools.

Use Distance Constraint Validator and Angle Constraint Validator to verify the constraint files used for this structure. You can work with integrated PyMOL and Sparky to validate the constraints (PyMOL, SPARKY installation needed). 

If you want to see it in PyMOL, click View in Space. It will bring up PyMOL. Once you have PyMOL launched by Ponderosa Analyzer, you do not need to re-launch. Just type @p in the PyMOL command to update the screen. After that, you can easily update by clicking on the up arrow and pressing “enter”.

PyMOL integration for validation

Spectral view in SPARKY is also supported. Click View in Spectrum, open your SPARKY project and type up two-letter code in SPARKY (NMRFAM-SPARKY required). Connect Ponderosa Analyzer window will pop up.

Connection file, sparky.cnt, will be set automatically. Set drop down menus for spectrum types. If you do not have specific NOESY, it can be set to any. Click the Update button to load the spectral position of your selection in your Distance and Angle Constraint Validator. Whenever you select a constraint in the Validator, click the Update button to reload the positions. You can move to the place of the constraint by double-clicking on the label or by clicking the Move button.

This will bring you to the position.

Keep validating violated constraints and correct them. 

[Distance Constraint Control] [Angle Constraint Control]

Use Constraint Controls to correct. Enable and Disable will make selected constraint effective and ineffective. Set to functions will change the value of constraints. For the example shown above, if you click Set to 5.0A, the upper limit will be changed to 5.0Å. In this way, you can fine-tune constraints.

A useful feature for tracking modifications is the Comment function. Double-clicking on a constraint will bring up the following dialog window.

You can add or edit any comment to the selected constraint.

By using this feature, you will not mess up your corrections.

After all the validation and correction are done, click Save in the Validator windows.

Click Export for Ponderosa Client and select a directory to write input constraints for Ponderosa Client. Exported file names will be postprocessed.upl, postprocessed.lol and postprocessed.aco.

Set up others as in the previous run in Ponderosa Client, but add additional constraints such as the following.

And do not forget setting Constraints only.

Click Analyze NOESY and Calculate strc for refined structure calculation.

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2. For Administrators

We provide the Ponderosa Server program to institutes that have valid CYANA licenses. The program is essential for PONDEROSA-C/S but not publicly available.

If you  decided running your own Ponderosa Server, please check  prerequisites first [Click here].

* Check STRIDE

$ stride

You must specify input file

Action: secondary structure assignment
Usage: stride [Options] InputFile [ > file ]
  -fFile      Output file
  -mFile      MolScript file
  -o          Report secondary structure summary Only
  -h          Report Hydrogen bonds
  -rId1Id2..  Read only chains Id1, Id2 ...
  -cId1Id2..  Process only Chains Id1, Id2 ...
  -q[File]    Generate SeQuence file in FASTA format and die

Options are position  and case insensitive

* Check TALOS-N (or others)

$ talosn
Failed to find an installed nmrPipe system
A stand-alone version TALOS-N installation is used.
   The following nmrPipe related features are disabled:
   . automatic TALOS/BMRB format input indentication and conversion
   . automatic identical database protein identification and exclusion

Testing /usr/local/src/talos_n/bin/TALOSN
Testing /usr/local/src/talos_n/bin/TALOSN.linux9_x64
Testing /usr/local/src/talos_n/bin/TALOSN.linux9
Testing /usr/local/src/talos_n/bin/TALOSN.linux
Running /usr/local/src/talos_n/bin/TALOSN.linux

* Check mail

$ mail
Mail version 8.1 6/6/93.  Type ? for help.
"/var/spool/mail/mani": 54 messages 54 new
>N  1 MAILER-DAEMON@zavot.  Sat Jul  2 23:06  98/3823  "Undelivered Mail Returne"

* Check CYANA

$ cyana
CYANA 3.0_intel (intel)
Copyright (c) 2002-08 Peter Guntert. All rights reserved.
    Library file "/usr/local/cyana-3.0_intel/lib/cyana.lib" read, 38 residue types.

* Check MolProbity

$ oneline-analysis
No input directory specified.

$ xplor
                                        Xplor-NIH version 2.35
 C.D. Schwieters, J.J.  Kuszewski,       Progr. NMR Spectr. 48, 47-62 (2006).
 N. Tjandra, and G.M. Clore              J. Magn. Res., 160, 66-74 (2003).
 http://nmr.cit.nih.gov/xplor-nih        based on X-PLOR 3.851 by A.T. Brunger

 User: whlee        on: aubrac.nmr(x86_64/Linux  )  at: 17-Apr-14 16:45:18

* Check CNS (not necessary if you have XPLOR-NIH installed)

$ cns

          |                                                          |
          |            Crystallography & NMR System (CNS)            |
          |                         CNSsolve                         |
          |                                                          |
           Version: 1.3
           Status: General release
           Written by: A.T.Brunger, P.D.Adams, G.M.Clore, W.L.DeLano,
                       P.Gros, R.W.Grosse-Kunstleve,J.-S.Jiang,J.M.Krahn,
                       J.Kuszewski, M.Nilges, N.S.Pannu, R.J.Read,
                       L.M.Rice, G.F.Schroeder, T.Simonson, G.L.Warren.
           Copyright (c) 1997-2010 Yale University
           Running on machine: xx.nmrfam.wisc.edu (x86_64/Linux,64-bit)
           Program started by: xxxxx
           Program started at: 10:56:10 on 14-Apr-2014

 FFT3C: Using FFTPACK4.1


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  1. Lee W, Petit CM, Cornilescu G, Stark JL, Markley JL. (2016) The AUDANA algorithm for automated protein 3D structure determination from NMR NOE data. Journal of Biomolecular NMR 65(2):51-57
  2. Lee W, Cornilescu G, Dashti H, Eghbalnia HR, Tonelli M, Westler WM, Butcher SE, Wildman-Henzler K, Markley JL. (2016) Integrative NMR for biomolecular research. Journal of Biomolecular NMR 64(4):307-332
  3. Lee W, Stark JL, Markley JL. (2014) PONDEROSA-C/S: Client-server based software package for automated protein 3D structure determination. Journal of Biomolecular NMR  60(2-3):73-5
  4. Lee W, Kim JH, Westler WM, Markley JL (2011) PONDEROSA, an automated 3D-NOESY peak picking program, enables automated protein structure determination. Bioinformatics 27: 1727–1728

Last Updated: October 26, 2018

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