Tutorial

InRegPep is an online web server for structural modeling of aggregates formed by amyloid-prone peptides that adopt an in-register, parallel β-sheet architecture. Note that this method is not designed to predict whether a given sequence forms amyloids; rather, it is intended to propose aggregate conformations for peptides that are already considered amyloidogenic.

Job submission

A job can be submitted using either Basic or Advanced mode.

Basic mode: The user provides only the amino acid sequence of an amyloidogenic peptide that forms aggregates (sequence length: 10–100 residues; format: uppercase letters only, using one-letter amino acid codes). After submission, the server generates a link to the results page. Optionally, the user may provide an email address, and the link to the result page will be sent by email. The user may also provide a name for the task in the Job name field. By default, modeling results are visible to everyone. To make it private and accessible only to the user who submitted the job, check the box (Check the box to hide results).

Advanced mode: The user can modify the following simulation parameters:

  1. Number of Docking Simulations: values from 50 to 100 are allowed (default: 50). For short peptides, 50 simulations are usually sufficient. For more complex systems (longer peptides), a higher number of runs is recommended. Increasing this value results in a longer docking trajectory and a larger model ensemble for analysis, but also increases the total job runtime on the server.
  2. Monte Carlo Steps: values from 50 to 100 are accepted (default: 50). For simple systems, 50 steps may be sufficient. Larger systems typically require more MC steps to achieve better sampling of the conformational space.
  3. Disulfide Bridges (-S–S-): allow the user to impose distance restraints on pairs of cysteine residues that mimic a disulfide bond. Use the format i:j (residue indices). For example, to define two disulfide bridges between residues 3 and 6 and between residues 11 and 16, enter: 3:6,11:16.
  4. Initial Temperature sets the simulated annealing temperature range (default: 2.0); values in the range 2.0–3.0 are accepted for the initial temperature.
  5. Final temperature: sets the simulated annealing temperature range (default 1.0); for initial and final temperatures, respectively); values in the range 1.0–1.5 are accepted for the final temperature.
  6. Secondary Structure: allows the user to define a secondary structure assignment for the submitted sequence (C = coil, E = extended). The default value is “C” for the entire peptide sequence. For example, to assign an extended conformation to the first 10 residues and a coil conformation to residues 11 to 15, enter: EEEEEEEEEECCCCC.
  7. pH: sets the pH value used to define protonation states of peptide amino acids during model reconstruction, molecular dynamics refinement, and scoring.

Running the Job

After job submission, a link to the job’s results page is generated. The results page becomes active once the job is completed. Calculations typically take from a few hours up to a few days, depending on the length of the submitted sequence and the cluster load.

If an email address was provided, a message containing a link to the results will be sent, along with a notification when the job is completed. The status of the submitted job can be viewed on the job list page: bioinfo.imdik.pan.pl/inregpep/joblist

Viewing the Results

After job completion, the results page becomes active. The simulation parameters and job name are listed at the top of the page.

A plot of CABS-dock energy vs. pcaRMSD is provided for each simulated system, illustrating the relationship between the energy of the forming aggregate and the translational symmetry of the aggregating peptides.

Structures and additional data for the 10 predicted aggregate models can be displayed using the buttons on the right (Model 1 to Model 10). For each model, the following data and analyses are provided:

  1. The 3D structure of the predicted aggregate (displayed in the panel: Models).
  2. A bar plot showing RMSD and pcaRMSD values: RMSD describes model conformational stability, while pcaRMSD reflects the translational symmetry of aggregating peptides. Both values are averaged over the final 1 ns of the MD simulation.
  3. A bar plot showing normalized β-sheet content and internal contacts between aggregating peptides, calculated over the entire MD simulation.
  4. A bar plot showing per-residue β-sheet content, calculated and normalized over the entire MD simulation.
  5. An RMSF plot showing residue-wise RMSF values calculated over the entire MD simulation; low RMSF values indicate regions of the aggregating peptide with lower conformational flexibility.
  6. A protofilament structure: for aggregate models ranked as plausible, a corresponding protofilament structure composed of 15 peptides is proposed.

All plots (graphics) and data (including structures) can be downloaded as a single ZIP archive by clicking "Download results." The archive also includes traj.zip - a compressed PDB-format trajectory of representative aggregate models in Cα-trace representation obtained from all docking trajectories. In particular, it includes the lowest-energy structure, the structure with the highest symmetry (as indicated by the pcaRMSD parameter), and 10 structures identified using k-means clustering for each Monte Carlo run. The total number of unique structures is equal to 12 × the number of Monte Carlo runs.