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name: rfdiffusion description: > Generate protein backbones using RFdiffusion, a diffusion-based generative model for de novo protein structure generation. Use this skill when: (1) Designing binder scaffolds for a target protein, (2) Generating novel protein backbones from scratch, (3) Scaffolding functional motifs into new proteins, (4) Specifying hotspot residues for interface design, (5) Creating symmetric oligomers.

For sequence design after backbone generation, use proteinmpnn. For structure validation, use alphafold or chai. For QC thresholds, use protein-qc. license: MIT category: design-tools tags: [structure-design, diffusion, backbone, binder] proteinbase_slug: rfdiffusion proteinbase_url: https://proteinbase.com/design-methods/rfdiffusion biomodals_script: modal_rfdiffusion.py

RFdiffusion Backbone Generation

Prerequisites

How to run

First time? See Installation Guide to set up Modal and biomodals.

Option 1: Modal (recommended)

Copy# Clone biomodals
git clone https://github.com/hgbrian/biomodals && cd biomodals

# Basic binder design
modal run modal_rfdiffusion.py \
  --pdb target.pdb \
  --contigs "A1-150/0 70-100" \
  --hotspot "A45,A67,A89" \
  --num-designs 100

# With custom GPU/timeout
GPU=A100 TIMEOUT=60 modal run modal_rfdiffusion.py \
  --pdb target.pdb \
  --contigs "A1-150/0 70-100" \
  --num-designs 100

GPU: A10G (24GB) | Timeout: 30min default

Option 2: Local installation

Copy# Clone and install
git clone https://github.com/RosettaCommons/RFdiffusion.git
cd RFdiffusion && pip install -e .

# Download weights
wget http://files.ipd.uw.edu/pub/RFdiffusion/models/Complex_base_ckpt.pt

# Run inference
python run_inference.py \
  inference.input_pdb=target.pdb \
  contigmap.contigs=[A1-150/0 70-100] \
  ppi.hotspot_res=[A45,A67,A89] \
  inference.num_designs=100

Config Schema (Hydra)

Contigmap Syntax

Copy# De novo single chain (50-100 residues)
contigmap.contigs=[50-100]

# Binder + target (A = target chain, fixed with /0)
contigmap.contigs=[A1-150/0 70-100]

# Motif scaffolding (preserve residues, /0 = fixed)
contigmap.contigs=[20-40/0 A10-30/0 20-40]

# Multi-chain binder
contigmap.contigs=[A1-100/0 B1-100/0 60-80]

# Variable length ranges
contigmap.contigs=[A1-150/0 50-100]  # Binder 50-100 AA

Hotspot Specification

Copy# Residues for interface (chain + resnum, no spaces)
ppi.hotspot_res=[A45,A67,A89]

Common mistakes

Contig Syntax

✅ Correct:

Copycontigmap.contigs=[A1-150/0 70-100]  # Target fixed (/0), binder variable

❌ Wrong:

Copycontigmap.contigs=[A1-150 70-100]    # Missing /0 - target will move!
contigmap.contigs="A1-150/0 70-100"  # Quotes break parsing
contigmap.contigs=[A1-150/0, 70-100] # Comma breaks parsing

Hotspot Residues

✅ Correct:

Copyppi.hotspot_res=[A45,A67,A89]        # Chain letter + residue number

❌ Wrong:

Copyppi.hotspot_res=[45,67,89]           # Missing chain letter
ppi.hotspot_res=[A45, A67, A89]      # Spaces break parsing
ppi.hotspot_res="A45,A67,A89"        # Quotes break parsing

Complete Parameter Reference

Core Parameters

Advanced Parameters

Symmetry Parameters

Fold Conditioning

Model Checkpoints

Common workflows

Binder Design

1. Prepare target PDB (trim to binding region + 10A buffer)
2. Identify 3-6 hotspot residues (exposed, conserved)
3. Generate 100-500 backbones
4. Pass to proteinmpnn for sequence design

Motif Scaffolding

1. Extract motif coordinates
2. Use /0 to fix motif in contigmap
3. Generate surrounding scaffold
4. Validate motif preservation (RMSD < 1.5A)

Symmetric Oligomers

Copy# C3 symmetric trimer
python run_inference.py \
  symmetry.symmetry=C3 \
  contigmap.contigs=[100-150] \
  inference.num_designs=50

# D2 symmetric tetramer
python run_inference.py \
  symmetry.symmetry=D2 \
  contigmap.contigs=[80-120] \
  symmetry.radius=25

# Supported symmetries: C2, C3, C4, C5, C6, D2, D3, D4, tetrahedral, octahedral

Partial Diffusion (Refinement)

Copy# Start from existing structure, diffuse from timestep 10
python run_inference.py \
  inference.input_pdb=initial.pdb \
  diffuser.partial_T=10 \
  contigmap.contigs=[A1-100]

Output format

Copyoutput/
├── output_0.pdb       # Generated backbone
├── output_1.pdb
├── ...
└── output_99.pdb

Each PDB contains polyalanine backbone - use proteinmpnn for sequence.

Sample output

Successful run

Copy$ python run_inference.py inference.input_pdb=target.pdb contigmap.contigs=[A1-150/0 70-100] inference.num_designs=100
[INFO] Loading model from Complex_base_ckpt.pt
[INFO] Generating design 1/100...
[INFO] Generating design 50/100...
[INFO] Generating design 100/100...
[INFO] Saved 100 designs to output/

Generated:
output/output_0.pdb (85 residues)
output/output_1.pdb (92 residues)
...

What good output looks like:

• File size: 3-8 KB per PDB (backbone only)
• Residue count within specified range
• Secondary structure visible in PyMOL (helices/sheets, not random coil)

Decision tree

CopyShould I use RFdiffusion?
│
├─ Need to generate protein backbone?
│  ├─ Yes → Continue below
│  └─ No, already have backbone → Use ProteinMPNN
│
├─ What type of design?
│  ├─ Binder for protein target → RFdiffusion ✓
│  ├─ De novo monomer → RFdiffusion ✓
│  ├─ Motif scaffolding → RFdiffusion ✓
│  └─ Symmetric assembly → RFdiffusion ✓
│
└─ Priority?
   ├─ Need highest success rate → Consider BindCraft
   ├─ Need diversity/exploration → RFdiffusion ✓
   └─ Need all-atom precision → Consider BoltzGen

Typical performance

Expected downstream yield: ~10-15% of backbones pass full QC after sequence design + validation.

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Verify

Copyls output/*.pdb | wc -l  # Should match num_designs

Troubleshooting

Designs lack secondary structure: Decrease noise_scale to 0.5-0.8 Binder not contacting hotspots: Verify residue numbering, increase num_designs OOM errors: Reduce batch size or use A100 GPU Slow generation: Reduce diffuser.T to 25-35

Error interpretation

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Next: proteinmpnn for sequence design → structure prediction for validation → protein-qc for filtering.