I recently did this for a covalently modified Cys. Follow these steps: 1. Generate a SMILES string for L-cysteine bonded the modification. Yours appears to be C1=CC=C(C=C1)C(=O)SCC(C(O)=O)N. https://pubchem.ncbi.nlm.nih.gov/compound/262741 2. Input the SMILES string to PHENIX eLBOW using the graphical user interface with quantum optimization enabled to generate restraints (.cif) and coordinates (.pdb). 3. Use the eLBOW graphical user interface to change the atom names for the backbone atoms of the modified cysteine to the standard ones for amino acids (N, CA, C, O, CB). 4. Save the new coordinates and restraints. 5. Move the modified cysteine you saved in step 4 into the density using Coot. 6. Delete the extra O atom of the cysteine carboxylate and save the coordinates of the modified Cys. 7. Using a text editor, change the residue number and chain ID of the modified cysteine to those of the Cys in your structure. Save the file. 8. Using a text editor, replace the Cys coordinates of your structure with the coordinates of the modified Cys save in step 7. In subsequent refinements, PHENIX refine should automatically incorporate the modified cysteine into the polypeptide chain, assuming you correctly renamed the atoms in step 3 and deleted the correct O atom in step 6. -- John J. Tanner Professor of Biochemistry and Chemistry Associate Chair of Biochemistry Department of Biochemistry University of Missouri 117 Schweitzer Hall 503 S College Avenue Columbia, MO 65211 Phone: 573-884-1280 Email: [email protected]mailto:[email protected] https://cafnrfaculty.missouri.edu/tannerlab/ Lab: Schlundt Annex rooms 3,6,9, 203B, 203C Office: Schlundt Annex 203A From: [email protected] on behalf of Sergio A. Sampieri Date: Friday, January 21, 2022 at 10:39 PM To: [email protected] Subject: [phenixbb] refining a covalent-bound ligand (part 2) WARNING: This message has originated from an External Source. This may be a phishing expedition that can result in unauthorized access to our IT System. Please use proper judgment and caution when opening attachments, clicking links, or responding to this email. Hi all, hope this message finds you well. As in a previous topic from 2018, I’m having problems refining a covalent-bounded ligand. The link shall be made between a carbon atom that is double bonded to an oxigen “SMILES C1=CC=C(C=C1)C=O” and the SG group of a Cys residue. I generated a CIF dictionary through AceDRG in COOT and CCP4i2. But after trying different aproaches during refinement, (Automatic Linking parameters, Atom selection, acceptor & donnor, ligand bond cut off and so) the result keeps being a non bounded enzyme-ligand complex (“Not linked: pdbres="LIG A 401”). I readed in the phenixbb that this was solved by changing the nature of the ligand (http://www.phenix-online.org/pipermail/phenixbb/2018-February/023718.htmlhttps://nam02.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.phenix-online.org%2Fpipermail%2Fphenixbb%2F2018-February%2F023718.html&data=04%7C01%7Ctannerjj%40missouri.edu%7C9ee2fb7d400b43ce4f2c08d9dd61150a%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C637784231772798225%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&sdata=jfdfpaNIDIvqF%2Foqqt1tlriMuWTRpL3%2F8CAz0oaVxS4%3D&reserved=0), but is not the way I would like to follow. Any suggestions or ideas would be greately appreciated, thanks a lot for your time. Stay safe.