Use RFdiffusion-v2

Official Neurosnap webserver for accessing RFdiffusion-v2 online.

Protein Design

Overview

RFdiffusion is an open source method for structure generation, with or without conditional information (a motif, target etc). It can perform a whole range of protein design challenges as outlined in the RFdiffusion paper.

Neurosnap Overview

The RFdiffusion-v2 online webserver allows anybody with a Neurosnap account to run and access RFdiffusion-v2, no downloads required. Information submitted through this webserver is kept confidential and never sold to third parties as detailed by our strong terms of service and privacy policy.

View Paper

Features

  • Motif Scaffolding
  • Unconditional protein generation
  • Symmetric unconditional generation (cyclic, dihedral and tetrahedral symmetries currently implemented, more coming!)
  • Symmetric motif scaffolding
  • Binder design
  • Design diversification ('partial diffusion', sampling around a design)

Configuration & Options

General Inputs and Settings

Allowed Types: pdb
The PDB structure of the protein you want to design a binder for, scaffold on, or partially diffuse. Please ensure this PDB file has chain IDs. An input PDB is REQUIRED for binder design, scaffolding, and partial diffusion.

Desired number of diffuser timesteps to generate your structure. Lower numbers produce lower quality results but have the advantage of taking less time. Recent improvements have allowed us to reduce the number of timesteps we need to use at inference time. In many cases, 20 steps provides outputs of equivalent quality to running with 200 steps (providing a 10X speedup). The default used by the RFdiffusion authors is now set to 50 steps, the former default was 200.

Check this option to use the "beta" model parameters for a better SSE balance. Useful if you are seeing lots of helices.

Binder Design Mode

Tips for designing binders like a pro:

Presenting the entire target, especially when it is large, like many cell-surface receptors, can significantly slow down the diffusion process. A well-established method to accelerate the design of binders involves cropping the target protein around the desired interaction site. This is typically done by defining a crop range using the Binding Pocket Residue Start and End. However, this approach poses a challenge: cropping might expose hydrophobic core residues that were previously buried, potentially misleading the binder to these new exposed areas rather than the intended interaction site on the target’s surface.

To address this issue, we suggest defining Hotspots that will bias the diffusion process to target those residues.

The target chain for the binder design experiment. This input is required if you are performing a scaffolding experiment.

The starting residue for the residues in range of your desired binding pocket target. If you are looking to design a binder for your input structure within range of residue 65 to 100, set this value to 65. To disable cropping set Binding Pocket Residue Start and Binding Pocket Residue End to 1.

The final residue for the residues in range of your desired binding pocket target. If you are looking to design a binder for your input structure within range of residue 65 to 100, set this value to 100. To disable cropping set Binding Pocket Residue Start and Binding Pocket Residue End to 1.

The minimum length of the designed binder. If you want your binder to be at least 10 residues long, set this value to 10.

The maximum length of the designed binder. If you want your binder to be at most 20 residues long, set this value to 20.

The model optionally readily learns that it should be making an interface which involving these hotspot residues. Input is ChainResidueNumber: A100 for residue 100 on chain A.

Motif Scaffolding Mode

The target chain for scaffolding. This input is required if you are performing a scaffolding experiment.

The minimum amount to extend the Input Structure by N-Terminally.

The maximum amount to extend the Input Structure by N-Terminally.

The minimum amount to extend the Input Structure by C-Terminally.

The maximum amount to extend the Input Structure by C-Terminally.

The starting residue of the scaffold. Residues between Scaffold Start and Scaffold End will be preserved and extended from. Set both Scaffold Start and Scaffold End to 1 in order to preserve the entire structure as a scaffold.

The ending residue of the scaffold. Residues between Scaffold Start and Scaffold End will be preserved and extended from. Set both Scaffold Start and Scaffold End to 1 in order to preserve the entire structure as a scaffold.

Partial Diffusion Mode

The target chain for partial diffusion. If you want to redesign the entire structure you may leave this blank.

Specify the number of noising steps (only used for the partial diffusion protocol).

The beginning of the sequence region you want to keep fixed. The rest of the sequence will be noised. If you want to fix residues 1 to 10 set this value to 1. If you want to redesign the entire structure you may leave this blank.

The end of the sequence region you want to keep fixed. The rest of the sequence will be noised. If you want to fix residues 1 to 10 set this value to 10. If you want to redesign the entire structure you may leave this blank.

Unconditional Protein Generation Mode

The desired length of the unconditionally generated protein. If you enter 100 the monomeric (order 1) protein will be 100 residues long. If you enter an order greater than 1 the protein will be that order multilpied by the length you specify.

Select the symmetrical design you want to use to predict your structure. Setting this to "auto" enables automatic symmetry dectection with AnAnaS.

Defines the number of copies in the symmetries. Think of it was the desired number of subunits in the predicted structure. This option will be ignored for Tetrahedral, Octahedral, and Icosahedral.

Potentials Settings

RFDiffusion can optionally take "guiding potentials" that can bias the diffusion process into designing structures with ideal properties. Potentials can be very powerful when used correctly and are primarily for those already comfortable with using RFDiffusion.

Key Tips For Potentials
  1. It is often good practice to start with no potentials as a baseline, then slowly increase their strength.
  2. Not all potentials are compatible with all RFdiffusion modes or even other potentials.
  3. Potentials for binder design, seem to interact weirdly with hotspot residues in PPI.
  4. Repetition and experimentation with different settings is key. If a combination of settings is consistently yielding poor results, try a new combination.

For more information on potentials we suggest this blog post.

Generates compact structures by minimizing the radius of gyration (ROG) of the Cα atoms. Can be only be used for unconditional generation, motif scaffolding, and partial diffusion.

Encourages the formation of contacts within a monomeric protein. Can only be used for unconditional generation, motif scaffolding, and partial diffusion.

Encourages the formation of contacts within a multimeric protein. Can only be used for unconditional symmetric generation.

Used for motif scaffolding, where the goal is to design a protein structure around a given functional motif. The potential mimics interactions between the designed protein and a virtual substrate or ligand, encouraging the formation of a binding site that accommodates the motif. This option requires a ligand / substrate to be included the uploaded structure.

User for designing protein binders. It encourages the model to generate compact structures for the binder region by minimizing the radius of gyration (ROG) of the Cα atoms in the binder. A smaller ROG indicates a more compact binder structure.

User for designing protein binders. Encourages the formation of contacts within the binder region and at the interface between the binder and the target protein, respectively. Favors structures with more contacts, which can contribute to the stability and specificity of the binder-target interaction. Can only be used for binder design.

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