1. What is PONDEROSA-C/S?

PONDEROSA-C/S is a new software package developed at NMRFAM (National Magnetic Resonance Facility at Madison) for automated protein 3D structure determination. PONDEROSA-C/S is a successor of the PONDEROSA program, which utilizes information from prior assignments to automatically pick NOE peaks from 3D-NOESY (13C-edited and 15N-edited) spectra and uses the results as input for a series of AUDANA (Automated Database-Assisted NOE Assignment) or CYANA structure calculations with the goal of achieving a publishable quality solution structure. Additionally, through-space data acquired from MAS (magic angle spinning) solid-state NMR spectroscopy are supported by development of AUDASA (Automated Database-Assisted Solid-State NMR Assignment). Compare to the original PONDEROSA, PONDEROSA-C/S offers improvements and many new innovations.

NMRFAM offers various software packages for high-throughput NMR. PONDEROSA-C/S is designed to interface with and extends other software packages such as, NMRFAM-SPARKY, PINE-NMR, PINE-SPARKY, ADAPT-NMR, and ADAPT-NMR Enhancer. The output from these packages is accepted as input to PONDEROSA-C/S, and, together, they offer an efficient pipeline for data collection, assignment, and structure determiantion.

PONDEROSA-C/S is designed to be accessible to persons who are not experts in NMR spectroscopy, and it offers a fast and easy way to determine protein 3D structures.

1. Resonance assignment file. NMR-STAR file format (.str), XEASY proton file format (.prot), PIPP file format (.pipp) or SPARKY assignment table format (.shifts).

2. Protein amino acid sequence file. In any one of three common formats: 3-letter-code with indices (.seq), FASTA 1-letter-code (.fasta), 3-letter or 1-letter in individual lines (.txt). Examples of each are given below.
   

• 3-letter-code with indices (.seq):

• MET  1
  THR  2
  ...
  

• FASTA format (.fasta):

• MTSRV.......

• 3-letter or 1-letter in individual lines (.txt):

• MET or M
  THR or T
  ...
  

3. 3D-NOESY (13C-edited and/or 15N-edited), 2D-NOESY and/or 2D-CC, 3D-NCOCX, 3D-NCACX from ssNMR: Both raw spectra and refined peak lists supported. Raw spectra should be in eother SPARKY (.ucsf) or NMRPipe (.pipe) format. Unrefined peak lists should be either SPARKY (.list) and XEASY (.peaks) format.
   

   • PONDEROSA-C/S has the capability of analyzing raw NMR spectra. If raw spectra are provided, PONDEROSA-C/S will carry out peak picking guided by the resonance assignments provided. If unrefined peak lists are provided as input, the peaks will be validated and refoned by PONDEROSA-C/S.

4. {Optional} Distance restraints (.upl, .lol), torsion angle restraints (.aco), residual dipolar couplings (.rdc), X-ray/cryo-em map (.map), blacklist/whitelist from Ponderosa Analyzer (.blt, .wlt), atomic coordinates .pdb or . cif), small angle X-ray scattering raw data (.dat).

OUTPUTS: Here is what you will get after a PONDEROSA-C/S run.

1. A family of conformers in PDB format that represent the protein 3D structure calculated by CYANA.

• Separated models in individual PDB format are also available.

2. 2D structure analysis, solvent accessible surface (SAS), torsion angles (PHI, PSI) from STRIDE and TALOS relatives (TALOS, TALOS+, TALOS-N).
   

3. MolProbity analysis file.

4. Lists of assigned and unassigned peak lists from the 3D-NOESY spectra in SPARKY and XEASY format.

5. Soon to be supported:

• COROM for refined torsion angle prediction.

2. What makes it so innovative?

NMR structures provide scientifically meaningful results, but NMR spectroscopy has been regarded as a harder and more expensive way of solving structures than X-ray crystallography. Thus, it is important to develop ways of making NMR more efficient and less expensive.

So, what makes PONDEROSA-C/S so innovative and distinctive?

1. It is automated.

   • Your protein structure will be determined in a day or two.

   • PONDEROSA-C/S manages all the input and output for each step.
     
   • Analyzed outputs from each step become input for the next step in the procedure.

   • Designed to work flawlessly with other automation packages.

     • NMRFAM packages: ADAPT-NMR, ADAPT-NMR Enhancer, PINE-NMR, PINE-SPARKY.

     • Other popular packages: TALOS versions, PALES, GNOM, DAMMIF, SPARKY, CYANA, Xplor-NIH.

2. Client-side and server-side are separated.
   

   • At the user side, the only software packages required are Ponderosa Client and Ponderosa Analyzer.

   • All the computationally intense jobs, such as structure calculation and analysis, are conducted in Ponderosa Server following data submission by the Ponderosa Client.

   • Honestly, SPARKY is required for validation against spectra and PyMOL is required for validation in 3D space, but most users will have these packages already.

   • If user wants to use PONDEROSA calculation runs with CYANA, and the one must either be NMRFAM collaborators to work under our license, or have an external installation of Ponderosa Server with your own CYANA license.
     

   • Outside users who wants to collaborate with NMRFAM and obtain support with a structure determianation should contact [Dr. Woonghee Lee] for detailed instructions. You will get your result back in 1-2 days after data submission.

   • Alternatively, Ponderosa Server can be installed on your site. The installation requires sufficient computational resources and some experience with computers. And you also need a valid licensed version of CYANA.

   • However, our new NOE assignment algorithm running with Xplor-NIH, called AUDANA (Automated Database-Assisted NOE Assignment) enables users to select either AUDANA automation or PONDEROSA-X refinement, and they are freely available. This algorithm uses RDC, SAXS and PONDEROSA Blacklists/Whitelists as auxiliary inputs, and we could find that this outruns CYANA automation in some challenging proteins.
     

3. Refinement tool for polishing your structure is provided.

• Ponderosa Analyzer is a dedicated tool for the analysis of PONDEROSA-C/S output.

• Ponderosa Analyzer works with the output from PONDEROSA-C/S output obtained by email.

• Ponderosa Analyzer provides an easy way of viewing structure quality scores.

• Ponderosa Analyzer provides tools integrated with PyMOL and SPARKY for easy structure refinement.

• Other tools are available for automated structure determination.
  

• With them, refinement has required a time-consuming iterative manual processes.

• Ponderosa Analyzer is unique in offering coordinated visualization of violations, constraints, and underlying spectra.

4. Supported by NMRFAM (National Magnetic Resonance Facility At Madison).

• PONDEROSA-C/S from NMRFAM always offers the most recent version of the software, which is under steady development. The server has the most recent updates and added new features.

• Plus, as long as you are collaborating with NMRFAM, we are ready to provide advice on any issues and obstacles.

3. Side-by-side comparison of the orignal PONDEROSA and PONDEROSA-C/S

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
   
• AUDANA automation and/or PONDEROSA-X refinement
  
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
   
• NMRFAM-SPARKY and/or Ponderosa Analyzer and/or Ponderosa Prediction Server and/or Video Tutorials
  
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
• Any software related to the PONDEROSA-C/S package
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
• PONDEROSA peak picking module (two-letter-code c3) in the NMRFAM-SPARKY

Last Updated: October 26, 2018