Tutorial: Using the HERCULES Web Server

Welcome to the HERCULES server. This service allows users to analyze RNA-binding ability of proteins at single amino acid resolution and perform mutagenesis analysis on individual protein sequences. This tutorial guides you through the full workflow, from data submission to result download.

Step-by-step Instructions

  1. Access the web server
    Open your web browser and navigate to the HERCULES website.
  2. Input your data
    You can provide input in one of the following ways:
    Option A: Upload a file
    • Click the Choose a file button.
    • Select a FASTA file from your computer (extension: .fasta).
    Option B: Enter a sequence
    • Scroll to the input window labeled Enter a sequence in FASTA format.
    • Type or paste your protein sequence in FASTA format.

    By default, HERCULES returns residue-level RNA-binding profiles and a global RNA-binding propensity score for the submitted protein(s).

    Optional: Mutagenesis analysis (single protein only)
    • If you submit one protein, you can enable mutagenesis analysis.
    • Check the box labeled Perform mutagenesis analysis.
  3. Enter your email (optional, highly recommended)
    Provide your email address to receive a notification with a direct link to your results once the analysis is completed.
    Recommendation: Adding an email is the best way to avoid losing your result link for long jobs.
  4. Submit the job
    Click Submit to start the analysis. You will be redirected to a confirmation page with a tracking ID and result link.
  5. Retrieve your results
    If you provided an email, you will receive a notification when your job is complete. Open the result link to view and download outputs.

What You Get in the Results

  • RNA-binding propensity scores for each submitted protein.
  • Residue-level RNA-binding propensity profiles in text format (with downloadable profile images in PNG).
  • If mutagenesis is enabled, a report with mutation scores for all possible amino acid substitutions, including a mutation heatmap by position and residue.

Expected Analysis Time

  • RBP propensity analysis is typically fast: about 1 second for one protein of 100 aa, and about 2 minutes for 100 proteins of around 100 aa.
  • Mutagenesis analysis takes longer and scales strongly with sequence length; as a reference, around 5 minutes for a 100 aa protein, with substantially longer times for larger proteins.

Troubleshooting

  • File upload issues: ensure files are valid FASTA and within the allowed size limits.
  • No results email: check spam/junk and verify your email address was entered correctly.
  • Error messages: follow the on-page guidance from the server, or contact support if the issue persists.
HERCULES workflow figure