Hi Claire, The recommended address for ChimeraX questions is chimerax-users@cgl.ucsf.edu <mailto:chimerax-users@cgl.ucsf.edu> CC'd here. Well, you have three protein structures, each opened from its own input file, so that's three models in ChimeraX. You can use the Join Models tab of Build Structure (in menu Tools... Structure Editing) to form a peptide bond between the C-term of one protein (in one model) and the N-term of another protein (in another model). That will make a new joined model. So you could do that twice -- the second time you would join the third protein chain to the previously joined first two. See help for details on the tool <https://rbvi.ucsf.edu/chimerax/docs/user/tools/buildstructure.html> <https://rbvi.ucsf.edu/chimerax/docs/user/tools/buildstructure.html#join> To use this tool you need to select specific backbone atoms to join. You can't see these atoms when the ribbon is shown, so you should hide ribbons and show atoms instead. There are several ways, but for example, these commands would show only the backbone atoms: preset sticks hide ~ @n,ca,c,o ...meaning use sticks preset, then hide atoms except those named N, C, CA, O (peptide backbone). How to specify atoms, residues, chains, ... in commands: <https://rbvi.ucsf.edu/chimerax/docs/user/commands/atomspec.html> Then you can Ctrl-click to select one atom, Shift-Ctrl-click to select a second atom, and then use the joining tool mentioned above. The selected atoms are shown with green outlines. However, first consider whether you need to delete any residues from any termini, and/or add residues like a longer linker. You can do that before the joining. There is a delete command, and the Actions menu has a choice to delete what is selected. If you need linker segments you can additional peptide models with Build Structure, Start Structure tab. <https://rbvi.ucsf.edu/chimerax/docs/user/commands/delete.html> <https://rbvi.ucsf.edu/chimerax/docs/user/tools/buildstructure.html#start> Then you would need additional joining steps to combine the other proteins with your linker(s). I hope this helps, Elaine ----- Elaine C. Meng, Ph.D. UCSF Chimera(X) team Department of Pharmaceutical Chemistry University of California, San Francisco

On Feb 20, 2025, at 8:31 AM, Nieder, Claire via ChimeraX-users <chimerax-users@cgl.ucsf.edu> wrote:

Hey there,

I am a grad student at UConn trying to covalently link two PDB models by inserting a GFP into the third intracellular loop of 5HT4. If you have any advice, I would really appreciate it.

Thanks, Claire Nieder Kienzler Lab UConn Chemistry

Hi Claire, You can open the structure twice and delete the second part from one copy and delete the first part from the other copy. Then you would have two models with the two parts. Elaine

On Feb 20, 2025, at 1:54 PM, Nieder, Claire via ChimeraX-users <chimerax-users@cgl.ucsf.edu> wrote:

This helps a lot. What if one of my models needs to be broken up into two models? I need to do this because the program does not recognize the new 'C' and 'N' terminus formed by the cutting of the third intracellular loop of a GPCR. Once this model is broken into two, the 'build join peptide' command should work.

Thanks, Claire Nieder

From: Elaine Meng <meng@cgl.ucsf.edu> Sent: Thursday, February 20, 2025 12:10 PM To: Nieder, Claire <claire.nieder@uconn.edu> Cc: ChimeraX Users Help <chimerax-users@cgl.ucsf.edu> Subject: Re: [chimerax-users] ChimeraX Model Joining *Message sent from a system outside of UConn.*

Hi Claire, The recommended address for ChimeraX questions is chimerax-users@cgl.ucsf.edu<mailto:chimerax-users@cgl.ucsf.edu> CC'd here.

Well, you have three protein structures, each opened from its own input file, so that's three models in ChimeraX.

You can use the Join Models tab of Build Structure (in menu Tools... Structure Editing) to form a peptide bond between the C-term of one protein (in one model) and the N-term of another protein (in another model). That will make a new joined model. So you could do that twice -- the second time you would join the third protein chain to the previously joined first two.

See help for details on the tool <https://nam10.safelinks.protection.outlook.com/?url=https%3A%2F%2Frbvi.ucsf....> <https://nam10.safelinks.protection.outlook.com/?url=https%3A%2F%2Frbvi.ucsf....>

To use this tool you need to select specific backbone atoms to join. You can't see these atoms when the ribbon is shown, so you should hide ribbons and show atoms instead. There are several ways, but for example, these commands would show only the backbone atoms:

preset sticks hide ~ @n,ca,c,o

...meaning use sticks preset, then hide atoms except those named N, C, CA, O (peptide backbone). How to specify atoms, residues, chains, ... in commands: <https://nam10.safelinks.protection.outlook.com/?url=https%3A%2F%2Frbvi.ucsf....>

Then you can Ctrl-click to select one atom, Shift-Ctrl-click to select a second atom, and then use the joining tool mentioned above. The selected atoms are shown with green outlines.

However, first consider whether you need to delete any residues from any termini, and/or add residues like a longer linker. You can do that before the joining. There is a delete command, and the Actions menu has a choice to delete what is selected. If you need linker segments you can additional peptide models with Build Structure, Start Structure tab. <https://nam10.safelinks.protection.outlook.com/?url=https%3A%2F%2Frbvi.ucsf....> <https://nam10.safelinks.protection.outlook.com/?url=https%3A%2F%2Frbvi.ucsf....>

Then you would need additional joining steps to combine the other proteins with your linker(s).

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

...

On Feb 20, 2025, at 8:31 AM, Nieder, Claire via ChimeraX-users <chimerax-users@cgl.ucsf.edu> wrote:

Hey there,

I am a grad student at UConn trying to covalently link two PDB models by inserting a GFP into the third intracellular loop of 5HT4. If you have any advice, I would really appreciate it.

Thanks, Claire Nieder Kienzler Lab UConn Chemistry

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