ColabDock

Protein-protein docking guided by experimental restraints using AlphaFold2

Job name

Proteins (Chain: A)

Proteins (Chain: B)

150

Configure input settings on the left, then click "Submit"

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What is ColabDock?

ColabDock is a protein-protein docking framework that integrates AlphaFold2 with experimental restraints to predict how proteins bind to each other. Developed by Feng and colleagues at Peking University, it was published in Nature Machine Intelligence in August 2024.

Unlike traditional docking methods that use Fast Fourier Transform (FFT) algorithms like ZDOCK, HADDOCK, or ClusPro, ColabDock uses gradient backpropagation to optimize docking poses. This approach automatically integrates the AlphaFold2 energy function with experimental data without requiring retraining.

ColabDock is particularly useful when you have experimental data about protein-protein interfaces—such as cross-linking mass spectrometry (XL-MS), NMR chemical shift perturbation, or covalent labeling experiments. The method outperforms HADDOCK and ClusPro on benchmarks using both simulated and real experimental restraints.

How does ColabDock work?

Generation stage

ColabDock operates in two stages. The generation stage uses ColabDesign, a protein design framework built on AlphaFold2, to create initial complex structures.

During generation, the model optimizes sequence representations in logit space while minimizing four loss functions: a monomer distogram loss (preserving individual chain conformations), a restraint loss (bringing specified residues close together), and pLDDT/ipAE losses (ensuring high-quality predictions). The weighted combination of these losses guides the model toward structures that satisfy both the input templates and experimental constraints.

Prediction stage

After generation, AlphaFold2 predicts the final complex structure using the generated conformation as a guide. This two-stage approach combines the flexibility of gradient-based optimization with AlphaFold2's accuracy.

Ranking algorithm

ColabDock ranks output structures using a RankingSVM model trained on five features: ipTM (interface quality), contact number, contact pLDDT, number of satisfied restraints, and average error. This ranking helps identify the most likely binding mode when multiple conformations are generated.

Input requirements

Protein inputs

ColabDock requires 2-4 protein chain structures as input. You can upload single-chain PDB files directly or fetch chain-specific structures from the RCSB database using identifiers such as 6W63:A.

Each primary protein card should represent one docking partner chain. For typical binary complexes, provide the two interacting chains. For larger assemblies, you can include up to four chains total, but each input must still be a single chain.

If you start from a multichain experimental structure, split it before docking or fetch the exact chain you need. For example, use 6W63:A and 5LU7:B as separate docking inputs instead of uploading the full 5LU7 tetramer as one partner.

Reference complex (optional)

If you have a known complex structure, you can upload it as an optional reference/native complex. ProteinIQ uses the upstream extract_rest.py helper from ColabDock to sample restraints from that structure, and the same reference can also be passed through as the native complex for optional RMSD reporting in the final ranking summary.

This is useful when you want faithful upstream restraint sampling from a known answer or when benchmarking docking poses against an experimentally determined complex.

Unlike the primary docking inputs, the optional reference complex can remain multichain. The important constraint is compatibility: the chain and residue numbering must already match the uploaded docking partners. ProteinIQ normalizes the first matching chains to A,B,C,... for upstream ColabDock, but it does not perform homolog mapping or residue renumbering.

Restraint types

Experimental restraints encode spatial relationships between residues—typically from cross-linking, NMR, or mutagenesis experiments. ColabDock supports four restraint formats with increasing flexibility.

1v1 restraints (specific pairs)

The simplest restraint type: two specific residues should be in contact. Format each restraint as chain:residue,chain:residue on separate lines.

Example: A:4,B:15 means residue 4 of chain A should contact residue 15 of chain B.

Use 1v1 restraints when you have precise residue-level data, such as identified cross-linked peptide pairs.

1vN restraints (one to range)

When you know one residue contacts somewhere within a region—but not the exact partner—use 1vN restraints. Format: chain:residue,chain:start-end or chain:residue,chain:residue,residue,....

Examples: A:4,B:13-18 means residue 4 of chain A contacts at least one residue between 13-18 of chain B. A:10,B:20,22,24-26 means residue 10 of chain A can satisfy the restraint by contacting residue 20, 22, 24, 25, or 26 of chain B.

This accommodates experimental uncertainty or lower-resolution data like NMR chemical shift perturbation that identifies affected regions rather than specific residues.

MvN restraints (multiple with minimum)

For ambiguous data where several restraints could be satisfied, MvN allows you to specify that only some must be true. Format: pair1;pair2;...|min_count.

Example: A:4,B:13-18;A:6,B:13,15,17|1 means at least one of the two candidate 1vN constraints must be satisfied.

This is valuable for noisy experimental data where some cross-links may be false positives.

Repulsive restraints (keep apart)

Some experiments reveal which regions do NOT interact. Repulsive restraints force specified residue pairs to remain distant. Format: chain:residue,chain:residue.

Example: A:6,B:18 means residue 6 of chain A should be far from residue 18 of chain B.

Use repulsive restraints when you have negative data—regions that definitively don't form the interface.

Prediction parameters

Optimization rounds: Number of generation-prediction cycles. Additional rounds can improve results for difficult cases.
Steps per round: Backpropagation steps during optimization. Most cases converge within 50 steps; increase for challenging targets.

Reference extraction options

When using a reference complex, these settings map directly onto the upstream extract_rest.py sampler:

Chains to sample between: The two normalized docking chains to sample restraints between, such as A,B.
Restraint type: Upstream sampling mode: 1v1, 1vN, MvN, or repulsive.
Number of sampled restraints: Upstream --num value.
1vN set size (N): Upstream --N value used for 1vN and MvN.
MvN count (M): Upstream --M value used when sampling MvN restraints.

Advanced options

Contact threshold: Upstream restraint distance cutoff in Angstroms. The 8.0 Å default is standard for cross-linking studies and is also passed to upstream restraint sampling.
Repulsive threshold: Minimum distance for repulsive restraints. Residues should be farther apart than this value.
Fixed chain groups: Optional upstream fixed_chains setting. Use semicolon-separated groups such as A,B;C,D to keep the relative positions within those groups fixed during docking.
Segment crop length: Optional upstream crop_len setting. This enables segment-based optimization for larger complexes when GPU memory is limiting.
Use AF2-Multimer: Enables the AlphaFold2-Multimer model, which is optimized for multi-chain prediction. We recommend keeping this enabled for most cases.

Understanding the results

Upstream ColabDock writes three artifact folders: gen, pred, and docked. ProteinIQ returns all of them in the Files tab. The docked folder contains the ranked top 5 docked complexes, while summary.txt records the upstream ipTM score, the number of satisfied restraints, and optional RMSD values when a reference/native complex was provided.

Examine the top 3-5 poses rather than relying solely on rank 1. Multiple similar top poses suggest a confident prediction, while diverse poses may indicate conformational flexibility or an uncertain binding mode.

If you provided restraints, check how many are satisfied in each pose. A good prediction should satisfy most attractive restraints while avoiding contacts flagged as repulsive. ipTM is used as the primary interface-quality score in the ProteinIQ data view, but the raw upstream summary.txt is also available for download.

Comparison to other docking tools

Tool	Approach	Restraint support	Best for
ColabDock	AF2 + gradient backpropagation	1v1, 1vN, MvN, repulsive	Data-driven docking with XL-MS/NMR
LightDock	Glowworm Swarm Optimization	Limited	Blind docking, flexibility
HADDOCK	FFT + refinement	Ambiguous restraints	NMR-driven docking
ClusPro	FFT + clustering	None	Blind docking screening

ColabDock is the preferred choice when you have experimental restraint data. For blind docking without prior knowledge, consider LightDock or ClusPro.

Limitations

ColabDock has a maximum restraint distance of 22 Å, determined by AlphaFold2's distance map upper limit. This restricts compatibility to shorter cross-linking reagents; longer-range XL-MS data cannot be directly incorporated.

The method can process complexes up to approximately 1,200 residues on an NVIDIA A100 GPU. Larger assemblies require segment-based optimization or splitting into subcomplexes.

Without experimental restraints, ColabDock may not outperform dedicated blind docking tools. The method's strength lies in integrating experimental data with deep learning predictions.