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PONDEROSA-C/S consists of three programs. User only needs to install Ponderosa Client for job submission and Ponderosa Analyzer for validation and refinement. Ponderosa Server only needs to be installed if you want to build your own server on your computational resources (many prerequisites needed with valid CYANA license). [Click here to move to the download section!]

1. Ponderosa Client

Ponderosa Client program is a tool for submitting your input to the Ponderosa Server.  This is a must-have program to run PONDEROSA-C/S even though you are thinking of manual refinment and are not planning to use Ponderosa Analyzer.

Your contact information must be filled in correctly to receive the results.  You will receive an e-mail notification that your job submission has been received by the server a couple of minutes after you click the “Calculate Strc” button. If this message is not received, it means that your submission failed to arrive or was denied by the server for some reason, so please contact the server administrator or Dr. Woonghee Lee.

Supported chemical shift file formats are XEASY prot (.prot), BMRB NMR-STAR (.str), SPARKY shift (.shifts), SPARKY resonance list (.list) and PIPP (.pipp). Ponderosa Client automatically generates an XEASY prot format file (.prot).

Many sequence file formats are supported by Ponderosa Client. General 3-letter-code with indices (.seq) pairs with XEASY prot is accepted. FASTA (.fasta), or 3-letter-code or 1-letter-code without indices in individual lines (.txt) are also supported. Ponderosa Client automatically generates 3-letter-code with indices format (.seq) from the other formats. For disulfide bonds and RDCs, Ponderosa Server automatically modifies the sequence file (CYS to CYSS, pseudo-linkages). Note that if you use .fasta or .txt, the first sequence index is assigned to 1.

Supported NOESY formats are two sorts: 1) raw NOESY spectra (.ucsf, .pipe , .nv or processed Bruker 3rrr), 2) unrefined peak lists (.peaks or .list). If the input is raw NOESY spectra, Ponderosa Client starts by identifying peaks and conducts an initial peak refinement. This takes seconds to minutes. Once it is completed, all the peaks saved in the working directory, and the process is not repeated whenever you change the settings or other input. This step is necessary because raw NOESY spectra require much more storage than peak lists. If peak lists are supplied instead of raw NOESY spectra, this step is skipped, and only the initial refinement filter will be applied.

Six types of constraints are supported: 1) torsion angle constraints (.aco), 2) distance constraints both upper limits (.upl) and lower limits (.lol), 3) residual dipolar couplings (.rdc), 4) SAXS raw data (.dat), 5) Ponderosa Blacklist (.blt), Whitelist (.wlt), and 6) coordinates of similar protein structure (.pdb). They are all optional. The Ponderosa Server will automatically generate .aco / .upl / .lol / .tbl files by use of PONDEROSA internal algorithms and/or TALOS / CYANA external modules. The user can provide hydrogen bond restraints in .upl and .lol formats.

Disulfide bonds will be considered automatically by Ponderosa Server, so please use regular CYS nomenclature not CYSS in your sequence file.

Weighting factors These are set to the default value of 50 for the initial run. If you think the resulting structures are biased, you can change them for subsequent runs.

Performance options can be set by the user in Ponderosa Client, but performance options in Ponderosa Server have higher priority.

There are eight assignment options: 1) CYANA automation: uses the CYANA NOESY assignment module to generate distance restraints and run structure calculation with them; 2) PONDEROSA refinement hacks into the CYANA automation and tries to remove violated constraints and recalculate the structure; 3) Constraints only: does not use any NOESY spectra or peak lists but only the distance constraints submitted by the user. This option is used followed by Ponderosa Analyzer in the final refinement step. 4) AUDANA automation: automated NOE assignment by AUDANA algorithm in Ponderosa Server and simulated annealing by Xplor-NIH, 5) PONDEROSA-X refinement: one additional Xplor-NIH structure calculation with refined constraints generated by AUDANA algorithm, 6) Constraints only-X: structure calculation for 40 structures (small scale for fast calculation) with only user uploaded constraints by XPLOR-NIH, 7) Constraints only-X for final step with implicit solvation: large number of structure calculation (100 structures) and simulataneous water refinement by XPLOR-NIH with EEFX potential, 8) Constraints only-X for final step with explicit solvation: large number of structure calculation (100 structures) and following explicit water refinement by XPLOR-NIH.

URL  is the location where the Ponderosa Server is installed. When you become a NMRFAM user, you will receive detailed instruction for utilizing our resources.

After submitting the input, you will need to run "Analyze NOESY" for initial peak picking and refinement. It will take seconds to minutes as mentioned above. "Calculate strc " causes the job to be submitted to the Ponderosa Server. After submission, you no longer need to keep Ponderosa Client open because all the jobs are carried out on the server side. "Review results" executes Ponderosa Analyzer.

Screen shot of Ponderosa Client program.


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    2. Ponderosa Server

    If your institution does not run Ponderosa Server on its site, you do not have to read this section. [Click here to jump to the Ponderosa Analyzer]

    To run and maintain Ponderosa Server program on your site, there are some prerequisites to consider.

    1. Operating system must be Linux. NMRFAM runs Ponderosa Server on the CentOS 6 version of Linux.

    2. Have a valid CYANA license, and make it run in any PATH by setting environmental PATH (optional for CYANA based calculations). Usually making a symbolic link to the /usr/local/bin will work. Alternatively (or in parallel), XPLOR-NIH Ver 2.35 or higher version can be used for Xplor-NIH based calculations.

    3. STRIDE and any of TALOS versions to be installed on server. They need to be set in environmental PATH, too.

    4. MOLPROBITY enhances the reliability and quality of results. Please install the stand-alone version from here.

    5. XPLOR-NIH Ver 2.35 or higher, or CNS Ver 1.3 is required to be installed on server with environment PATH setting for water refinement.

    6. Set up mail. This mailer will send notifications to the users and administrator.

    7. If you have multiple servers to equally distribute jobs, you need to set up NFS (Network File System) to share the files at the same directory names.

    Here are the settings of Ponderosa Server program:

    1. Upload directory is where all the data are saved and shared through NFS. Results will be under this directory, too.

    2. External directory is where your apache server allows external access. It will be seen as External URL from outside.

    3. In the External directory contains text files to be sent by the e-mail module to users and administrator. "welcome_email.txt" will be sent when the job is submitted at first, "success_email.txt" and "failure_email.txt" will be sent when the job is succeeded or failed. "admin_email.txt" contains administrator's email address. They will be automatically created when Ponderosa Server is run for the first time.


    Screen shot of Ponderosa Server program.


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    3. Ponderosa Analyzer

    Ponderosa Analyzer is a tool to validate and refine your structure produced by Ponderosa Server. You will receive a compressed zip file after a few minutes to hours after starting the run. PONDEROSA-C/S provides a lot of information regarding your structure, and you may not understand all the directory hierarchies and files. Ponderosa Analyzer is designed to show the important information in the easiest manner for users and provides tools for refinement to achieve a robust, violation-free. publishable structure.

    After unzipping the output on your local folder, load the working directory into Ponderosa Analyzer. If it is the first time you are working on the output, it will take a minute for Ponderosa Analyzer to make a complete backup in case you want to go back to the first output from Ponderosa Server.

    The grid will show calculation directories in each line, and they are categorized by four stages: 1) Best Evaluated (B), 2) Refined (R), 3) Draft (D), 4) Constraints only. Best Evaluatedis the structure with the best score (minimum value) obtained by the Ponderosa Server scoring system. The score includes various factors and so does not necessarily agree with individual factors such as the MolProbity score or target function score. Refined and Draft are about NOESY peak refinement steps during regulating noise threshold for NOESY. You can refer the description of CYCLES and STEPS in the original PONDEROSA document. Constraints only will be present if you used distance constraints as inputs rather than NOESY data. Each line is selectable in the grid, and you can check and correct distance and angle violations by clicking “Show Validation Tools”.

    After the correction, you can export your post-processed distance and angle constraints for a Constraints only job submission from Ponderosa Client.

    Screen shot of Ponderosa Analyzer program.


    Screen shot of Distance Constraint Validator from Ponderosa Analyzer.


    Screen shot of Angle Constraint Validator from Ponderosa Analyzer.


    Ponderosa Violation Investigator

    Ponderosa VI (Ponderosa Violation Investigator) is a simplified distance/angle constraint validation program that allows you to investigate violations reported from Ponderosa Server calculation. By integrating the program to PyMOL, user can see selected constraints (multi-selection allowed) in the PyMOL viewer.


    4. Download

    We provide the PONDEROSA-C/S packages. Ponderosa Client and Ponderosa Analyzer in binary executable formats for Linux (tested on Ubuntu 12.04+ and CentOS 5.5+ with QT 4.8+), Windows and MacOSX (Snow Leopard or newer). Ponderosa Server is in binary executable format for Linux only (tested on Ubuntu 12.04+ and CentOS 5.5+ with QT 4.8+).

    * Download

    Ponderosa Client [Linux] [Windows] [MacOSX] 

    Ponderosa Analyzer [Linux] [Windows] [MacOSX] 

    Ponderosa Server [Linux only] 

    Ponderosa VI [Linux] [Windows] [MacOSX] 

    * Installation

    Simply, unpack downloaded zip file to the convenient place and enjoy!

    If it complains about libraries or crashes, just install QT libraries (search for libQtGui) for Ponderosa Client and Ponderosa Server, and Lazarus libraries for Ponderosa Analyzer including GDK-PixBuf libraries (search for libgdk-pixbuf).

    * Extended development on this software package


    If you want to develop with your novel idea onto our package, contact Prof. John Markley or Dr. Woonghee Lee for details and source codes.

    P.S. Old original PONDEROSA is still downloadable from http://ponderosa.sourceforge.net. However, we highly recommend using the new PONDEROSA-C/S version.

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    [Jump to the Ponderosa VI]


    Reference:
    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: September 29, 2017
    By Woonghee Lee (whlee@nmrfam.wisc.edu)


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