Hi Jernej, I can offer some thoughts generically, but I leave it up to you to decide if any make sense in the context of what you already know about this system! Maybe others on this list with more experience predicting multimeric structures can supplement this reply. (1) Evaluate the quality of the predicted interactions between the parts, primarily by looking at the residue-residue PAE plot if you have it available. <https://rbvi.ucsf.edu/chimerax/docs/user/tools/alphafold.html#pae> <https://www.rbvi.ucsf.edu/chimerax/data/pae-apr2022/pae.html> (2) Consider any experimental evidence you have on this system as to which residues of the two parts may be in contact. Is the prediction consistent with this information? (3) To get more possible predictions, you could try additional methods, namely: - AlphaFold 3 (if possible) if what you already tried was AlphaFold 2, and vice versa. The ChimeraX interface only runs the ColabFold version based on AlphaFold 2 <https://rbvi.ucsf.edu/chimerax/docs/user/tools/alphafold.html> ... but maybe the separate AlphaFold 3 web server allows this kind of calculation. I am not sure. - Boltz, which is somewhat based on AlphaFold. Currently there is a ChimeraX interface to installing and running Boltz-1 on your own system. There is a Boltz-2 release, but ChimeraX does not have an interface to it at this time. <https://rbvi.ucsf.edu/chimerax/docs/user/tools/boltz.html> Some other thoughts are: - depending on how large a rearrangement you expect, you might try predicting the pre-cleaved single sequence and see if that also makes sense in the light of any experimental evidence as well as the predicted structure(s) of the cleaved heterodimer. Maybe it's not relevant, or maybe it would aid your understanding of this system somehow. Regarding making a tetramer from a dimer, ChimeraX does have a "sym" command for using specified symmetry to multimerize a structure. However, that means you would need to know the symmetry already -- it is not going to predict the dimer-dimer interface for you. <https://rbvi.ucsf.edu/chimerax/docs/user/commands/sym.html> Are there known homologous tetramer structures? If so you could open the known tetramer and superimpose two copies of your predicted dimer onto it, e.g. with ChimeraX's Matchmaker tool. <https://rbvi.ucsf.edu/chimerax/docs/user/tools/matchmaker.html> I hope this helps, Elaine ----- Elaine C. Meng, Ph.D. UCSF Chimera(X) team Resource for Biocomputing, Visualization, and Informatics Department of Pharmaceutical Chemistry University of California, San Francisco

On Jun 18, 2025, at 3:41 PM, Jernej Turnsek via ChimeraX-users <chimerax-users@cgl.ucsf.edu> wrote:

Hi All, I am working on a bifunctional enzyme with these two hypothesized features:

- it is first autocatalytically cleaved to form a heterodimer; - it then forms a homotetramer of heterodimers.

These two events would lead to a protein assembly with 2 active sites for catalytic activity A and 1 active site for catalytic activity B.

I have two questions:

1. I have modelled the initial heterodimer by "cleaving" the preprotein at the predicted autocatalytic cleavage site and feeding the resulting two aa sequences to the AlphaFold Server. Is there a better way to predict heterodimerization of such proteins using AlphaFold? 2. Is there a way to tetramerize the resulting AlphaFold heterodimer from 1. in ChimeraX?

Thank you and best wishes, Jernej

Thank you very much, Elaine and Tom! These are all very helpful tips. I am looking forward to diving deeper. Best wishes, Jernej On Thu, Jun 19, 2025 at 7:41 AM Tom Goddard <goddard@sonic.net> wrote:

Hi Jernej,

Cleaving the protein to make 2 sequences and predicting the heterodimer seems reasonable. Then you want to look at the Alphafold confidence in ChimeraX. Here is what to look at

https://www.rbvi.ucsf.edu/chimerax/data/pae-apr2022/pae.html

For the tetramer of heterodimers you could predict the tetramer using the Alphafold 3 server which can handle large complexes

https://deepmind.google/science/alphafold/alphafold-server/

Then look at the confidence of that prediction.

Tom

On Jun 18, 2025, at 8:55 PM, Elaine Meng via ChimeraX-users < chimerax-users@cgl.ucsf.edu> wrote:

Hi Jernej, I can offer some thoughts generically, but I leave it up to you to decide if any make sense in the context of what you already know about this system!

Maybe others on this list with more experience predicting multimeric structures can supplement this reply.

(1) Evaluate the quality of the predicted interactions between the parts, primarily by looking at the residue-residue PAE plot if you have it available. <https://rbvi.ucsf.edu/chimerax/docs/user/tools/alphafold.html#pae> <https://www.rbvi.ucsf.edu/chimerax/data/pae-apr2022/pae.html>

(2) Consider any experimental evidence you have on this system as to which residues of the two parts may be in contact. Is the prediction consistent with this information?

(3) To get more possible predictions, you could try additional methods, namely:

- AlphaFold 3 (if possible) if what you already tried was AlphaFold 2, and vice versa. The ChimeraX interface only runs the ColabFold version based on AlphaFold 2 <https://rbvi.ucsf.edu/chimerax/docs/user/tools/alphafold.html> ... but maybe the separate AlphaFold 3 web server allows this kind of calculation. I am not sure.

- Boltz, which is somewhat based on AlphaFold. Currently there is a ChimeraX interface to installing and running Boltz-1 on your own system. There is a Boltz-2 release, but ChimeraX does not have an interface to it at this time. <https://rbvi.ucsf.edu/chimerax/docs/user/tools/boltz.html>

Some other thoughts are:

- depending on how large a rearrangement you expect, you might try predicting the pre-cleaved single sequence and see if that also makes sense in the light of any experimental evidence as well as the predicted structure(s) of the cleaved heterodimer. Maybe it's not relevant, or maybe it would aid your understanding of this system somehow.

Regarding making a tetramer from a dimer, ChimeraX does have a "sym" command for using specified symmetry to multimerize a structure. However, that means you would need to know the symmetry already -- it is not going to predict the dimer-dimer interface for you. <https://rbvi.ucsf.edu/chimerax/docs/user/commands/sym.html>

Are there known homologous tetramer structures? If so you could open the known tetramer and superimpose two copies of your predicted dimer onto it, e.g. with ChimeraX's Matchmaker tool. <https://rbvi.ucsf.edu/chimerax/docs/user/tools/matchmaker.html>

I hope this helps, Elaine ----- Elaine C. Meng, Ph.D. UCSF Chimera(X) team Resource for Biocomputing, Visualization, and Informatics Department of Pharmaceutical Chemistry University of California, San Francisco

On Jun 18, 2025, at 3:41 PM, Jernej Turnsek via ChimeraX-users < chimerax-users@cgl.ucsf.edu> wrote:

Hi All,

I am working on a bifunctional enzyme with these two hypothesized features:

- it is first autocatalytically cleaved to form a heterodimer;

- it then forms a homotetramer of heterodimers.

These two events would lead to a protein assembly with 2 active sites for catalytic activity A and 1 active site for catalytic activity B.

I have two questions:

1. I have modelled the initial heterodimer by "cleaving" the preprotein at the predicted autocatalytic cleavage site and feeding the resulting two aa sequences to the AlphaFold Server. Is there a better way to predict heterodimerization of such proteins using AlphaFold?

2. Is there a way to tetramerize the resulting AlphaFold heterodimer from 1. in ChimeraX?

Thank you and best wishes,

Jernej

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