Inquiry regarding protonation state sampling using Rosetta pH-mode with Relax protocol

[wangsuhaoshabi]

Dear Rosetta Commons Team

I am writing to seek your expert guidance on modeling protein protonation states at specific pH values using Rosetta.

I am currently working with a protein of 206 residues. My objective is to accurately determine its protonation states under specific pH conditions. My current plan is to utilize the relax protocol (specifically FastRelax) while enabling the pH_mode flags (e.g., -pH:pH_mode true, -pH:value_pH [value], and -set_weights e_pH 1.0).

I would like to ask for your advice on the following:

Feasibility: Is the relax protocol combined with pH_mode the most appropriate approach for a protein of this size (206 residues) to achieve realistic protonation states?

Sampling Depth: How many output structures (nstruct) would you recommend to ensure the protonation states have reached convergence and the results are statistically reliable?

Alternative Protocols: Are there other specialized protocols (such as rosetta_pka or specific pH-aware docking applications) that might be better suited for this purpose than standard Relax?

Thank you very much for your time and assistance. I look forward to your professional recommendations.

Best regards,
wangwen

[roccomoretti]

The pH mode of Rosetta hasn't seen much development outside of it's original introduction, so the best resources for best practices are the original paper and the two docking follow-ups.