# PRODIGY Nextflow Pipeline

A Nextflow pipeline for predicting binding affinity of protein-protein complexes using PRODIGY (PROtein binDIng enerGY prediction).

## Overview

PRODIGY is a contact-based method for predicting the binding affinity of protein-protein complexes from their 3D structures. This pipeline containerizes PRODIGY using Docker and orchestrates execution through Nextflow, enabling reproducible, scalable analysis of protein-protein interactions.

### Key Features

- **Automated binding affinity prediction** from PDB/mmCIF structures
- **Batch processing** of multiple protein complexes
- **Docker containerization** for reproducibility
- **Configurable parameters** for distance cutoffs, temperature, and chain selection
- **Optional outputs** including contact lists and PyMOL visualization scripts

## Scientific Background

PRODIGY predicts binding affinity by analyzing intermolecular contacts (ICs) at protein-protein interfaces. The method:

1. Identifies residue-residue contacts within a distance threshold (default: 5.5 Å)
2. Classifies contacts by residue type (charged, polar, apolar)
3. Analyzes the non-interacting surface (NIS) composition
4. Predicts binding free energy (ΔG) and dissociation constant (Kd)

The 5.5 Å distance cutoff was optimized to capture various non-bonded interactions including salt bridges, hydrogen bonds, and hydrophobic contacts.

## Requirements

### Software Dependencies

- [Nextflow](https://www.nextflow.io/) (≥21.04.0)
- [Docker](https://www.docker.com/) (≥20.10) or [Singularity](https://sylabs.io/singularity/) (≥3.0)

### Hardware Requirements

- CPU: 1+ cores per process
- Memory: 4 GB minimum recommended
- Storage: ~2 GB for Docker image

## Installation

### 1. Clone or Download the Pipeline

```bash
# Create pipeline directory
mkdir -p /path/to/prodigy_pipeline
cd /path/to/prodigy_pipeline

# Copy pipeline files (Dockerfile, main.nf, nextflow.config, params.json)
```

### 2. Build the Docker Image

```bash
docker build -t prodigy:latest .
```

### 3. Verify Installation

```bash
# Test Docker image
docker run --rm prodigy:latest prodigy --help

# Test Nextflow
nextflow run main.nf --help
```

## Usage

### Basic Usage

```bash
# Run on a single PDB file
nextflow run main.nf --pdb /path/to/complex.pdb --outdir /path/to/output

# Run on multiple PDB files
nextflow run main.nf --pdb '/path/to/structures/*.pdb' --outdir /path/to/output
```

### With Custom Parameters

```bash
nextflow run main.nf \
    --pdb '/path/to/structures/*.pdb' \
    --outdir /path/to/output \
    --distance_cutoff 5.5 \
    --acc_threshold 0.05 \
    --temperature 37.0 \
    --contact_list true \
    --pymol_selection true
```

### Chain Selection for Complex Interfaces

For antibody-antigen complexes or multi-chain proteins:

```bash
# Contacts between chains A and B only
nextflow run main.nf --pdb complex.pdb --selection 'A B'

# Heavy (H) and Light (L) chains as one molecule vs Antigen (A)
nextflow run main.nf --pdb antibody_antigen.pdb --selection 'H,L A'

# Three-way interface calculation
nextflow run main.nf --pdb complex.pdb --selection 'A B C'
```

### Using Singularity

```bash
nextflow run main.nf -profile singularity --pdb /path/to/complex.pdb
```

## Parameters

### Required Parameters

| Parameter | Description | Default |
|-----------|-------------|---------|
| `--pdb` | Path to input PDB/mmCIF file(s). Supports glob patterns. | `/mnt/OmicNAS/private/old/olamide/Prodigy/input/*.pdb` |
| `--outdir` | Output directory for results | `/mnt/OmicNAS/private/old/olamide/Prodigy/output` |

### Analysis Parameters

| Parameter | Description | Default | Range |
|-----------|-------------|---------|-------|
| `--distance_cutoff` | Distance threshold (Å) for defining intermolecular contacts | `5.5` | 1.0 - 20.0 |
| `--acc_threshold` | Relative accessibility threshold for surface residue identification | `0.05` | 0.0 - 1.0 |
| `--temperature` | Temperature (°C) for Kd calculation | `25.0` | -273.15 - 100.0 |
| `--selection` | Chain selection for interface calculation | `''` (all chains) | See examples |

### Output Control Parameters

| Parameter | Description | Default |
|-----------|-------------|---------|
| `--contact_list` | Generate detailed contact list file | `false` |
| `--pymol_selection` | Generate PyMOL visualization script | `false` |
| `--quiet` | Output only affinity values (minimal output) | `false` |

## Output Files

### Standard Output

For each input structure `<name>.pdb`, the pipeline generates:

| File | Description |
|------|-------------|
| `<name>_prodigy.txt` | Main results file with binding affinity prediction |

### Optional Output (when enabled)

| File | Description | Parameter |
|------|-------------|-----------|
| `<name>_contacts.txt` | List of all interface contacts | `--contact_list true` |
| `<name>_interface.pml` | PyMOL script for interface visualization | `--pymol_selection true` |

### Example Output

```
[!] Structure contains gaps:
    E ILE16 < Fragment 0 > E ALA183
    E TYR184 < Fragment 1 > E GLY187

[+] Executing 1 task(s) in total
##########################################
[+] Processing structure 1ppe_model0
[+] No. of intermolecular contacts: 86
[+] No. of charged-charged contacts: 5.0
[+] No. of charged-polar contacts: 10.0
[+] No. of charged-apolar contacts: 27.0
[+] No. of polar-polar contacts: 0.0
[+] No. of apolar-polar contacts: 20.0
[+] No. of apolar-apolar contacts: 24.0
[+] Percentage of apolar NIS residues: 34.10
[+] Percentage of charged NIS residues: 18.50
[++] Predicted binding affinity (kcal.mol-1):    -14.7
[++] Predicted dissociation constant (M) at 25.0˚C:  1.6e-11
```

### Output Interpretation

| Metric | Description |
|--------|-------------|
| **Intermolecular contacts** | Total number of residue-residue contacts at interface |
| **Contact types** | Breakdown by residue character (charged/polar/apolar) |
| **NIS residues** | Composition of non-interacting surface |
| **Binding affinity (ΔG)** | Predicted free energy of binding (kcal/mol). More negative = stronger binding |
| **Dissociation constant (Kd)** | Predicted Kd at specified temperature. Lower = tighter binding |

### Binding Affinity Scale

| ΔG (kcal/mol) | Kd (M) | Binding Strength |
|---------------|--------|------------------|
| -6 to -8 | 10⁻⁵ to 10⁻⁶ | Moderate |
| -8 to -10 | 10⁻⁶ to 10⁻⁷ | Strong |
| -10 to -12 | 10⁻⁷ to 10⁻⁹ | Very Strong |
| < -12 | < 10⁻⁹ | Extremely Strong |

## Test Data

Download example protein complexes from the RCSB PDB:

```bash
# Create input directory
mkdir -p /mnt/OmicNAS/private/old/olamide/Prodigy/input

# Download test structures
wget -O /mnt/OmicNAS/private/old/olamide/Prodigy/input/3bzd.pdb https://files.rcsb.org/download/3BZD.pdb
wget -O /mnt/OmicNAS/private/old/olamide/Prodigy/input/2oob.pdb https://files.rcsb.org/download/2OOB.pdb
wget -O /mnt/OmicNAS/private/old/olamide/Prodigy/input/1ppe.pdb https://files.rcsb.org/download/1PPE.pdb
```

### Expected Results

| Structure | Description | Expected ΔG (kcal/mol) |
|-----------|-------------|------------------------|
| 3BZD | Protein-protein complex | -9.4 |
| 2OOB | Protein-protein complex | -6.2 |
| 1PPE | Trypsin-inhibitor complex | -14.7 |

## Pipeline Structure

```
prodigy_pipeline/
├── Dockerfile          # Docker image definition
├── main.nf             # Nextflow pipeline script
├── nextflow.config     # Pipeline configuration
├── params.json         # Parameter documentation
└── README.md           # This file
```

## Docker Image Details

The Docker image is based on Python 3.12 and includes:

- **prodigy-prot** (v2.4.0) - Main PRODIGY package
- **biopython** (≥1.80) - PDB structure parsing
- **freesasa** (≥2.2.1) - Solvent accessible surface area calculation
- **numpy** (≥2) - Numerical computations

### Building the Image

```bash
docker build -t prodigy:latest .
```

### Running Standalone

```bash
# Run PRODIGY directly
docker run --rm -v /path/to/data:/data prodigy:latest prodigy /data/complex.pdb

# Get help
docker run --rm prodigy:latest prodigy --help
```

## Troubleshooting

### Common Issues

**1. Docker Hub Rate Limit Error**
```
ERROR: toomanyrequests: You have reached your pull rate limit
```
Solution: Log in to Docker Hub with `docker login` or wait and retry.

**2. Structure Contains Gaps Warning**
```
[!] Structure contains gaps
```
This is informational, not an error. PRODIGY handles missing residues automatically.

**3. No Intermolecular Contacts Found**
- Verify the structure contains multiple chains
- Check chain selection parameters
- Ensure chains are in contact (within distance cutoff)

**4. Permission Denied Errors**
```bash
# Run with user permissions
docker run --rm -u $(id -u):$(id -g) -v /path/to/data:/data prodigy:latest prodigy /data/complex.pdb
```

### Getting Help

```bash
# PRODIGY help
docker run --rm prodigy:latest prodigy --help

# Nextflow pipeline help
nextflow run main.nf --help
```

## Citation

If you use this pipeline, please cite the following publications:

### PRODIGY Method

1. **Xue LC, Rodrigues JP, Kastritis PL, Bonvin AM, Vangone A.** (2016)
   PRODIGY: a web server for predicting the binding affinity of protein-protein complexes.
   *Bioinformatics*, 32(23):3676-3678.
   [DOI: 10.1093/bioinformatics/btw514](https://doi.org/10.1093/bioinformatics/btw514)

2. **Vangone A, Bonvin AM.** (2015)
   Contacts-based prediction of binding affinity in protein-protein complexes.
   *eLife*, 4:e07454.
   [DOI: 10.7554/eLife.07454](https://doi.org/10.7554/eLife.07454)

3. **Kastritis PL, Rodrigues JP, Folkers GE, Boelens R, Bonvin AM.** (2014)
   Proteins feel more than they see: Fine-tuning of binding affinity by properties of the non-interacting surface.
   *Journal of Molecular Biology*, 426(14):2632-2652.
   [DOI: 10.1016/j.jmb.2014.04.017](https://doi.org/10.1016/j.jmb.2014.04.017)

### Software Dependencies

- **Nextflow**: Di Tommaso P, et al. (2017) Nextflow enables reproducible computational workflows. *Nature Biotechnology*, 35:316-319.
- **Biopython**: Cock PJ, et al. (2009) Biopython: freely available Python tools for computational molecular biology and bioinformatics. *Bioinformatics*, 25(11):1422-1423.
- **FreeSASA**: Mitternacht S. (2016) FreeSASA: An open source C library for solvent accessible surface area calculations. *F1000Research*, 5:189.

## License

This pipeline is distributed under the Apache License 2.0, consistent with the PRODIGY software license.

## Links

- **PRODIGY Web Server**: [https://wenmr.science.uu.nl/prodigy/](https://wenmr.science.uu.nl/prodigy/)
- **PRODIGY GitHub**: [https://github.com/haddocking/prodigy](https://github.com/haddocking/prodigy)
- **BonvinLab**: [https://www.bonvinlab.org/](https://www.bonvinlab.org/)
- **Nextflow**: [https://www.nextflow.io/](https://www.nextflow.io/)

## Support

For questions about:
- **PRODIGY method**: Contact the BonvinLab team at [ask.bioexcel.eu](https://ask.bioexcel.eu/)
- **This pipeline**: Open an issue in the repository

---

*Pipeline version: 2.4.0 | Last updated: January 2026*