Hi Byron, pdb is a plaintext file and it has strict rules about what information goes in what column of the file. For example residue type must go in columns 18-20. You can google for more info on pdb format specifications if you like. But in your case, there is for some reason a misalignment at the cysteine atoms:

ATOM   1324  CG2 VAL D 145      -1.412 -45.504 -39.926  1.00 63.63           C

ATOM   1325  H   VAL D 145      -4.587 -46.874 -39.579  1.00 68.04           H

TER    5057      VAL D 145

HETATM   1326  N   AKG D 146      -3.172 -48.663 -42.861  1.00 57.79           N

HETATM   1327  C5  AKG D 146      -3.325 -49.926 -43.595  1.00 60.48           C

HETATM   1328  C   AKG D 146      -4.345 -50.910 -43.015  1.00 57.22           C

HETATM   1329  O   AKG D 146      -5.534 -50.795 -43.265  1.00 54.67           O

HETATM   1330  C6  AKG D 146      -1.965 -50.574 -43.892  1.00 69.38           C

HETATM   1331  H   AKG D 146      -2.831 -47.883 -43.403  1.00 57.79           H

HETATM   15  O3  AKG D 146      -1.290 -53.701 -43.091  1.00 20.00           O

HETATM   16  C7  AKG D 146      -1.596 -53.313 -44.191  1.00 20.00           C

HETATM   17  S1  AKG D 146      -1.629 -51.552 -44.626  1.00 20.00           S

HETATM   18  C8  AKG D 146      -1.983 -54.139 -45.371  1.00 20.00           C

HETATM   19  C9  AKG D 146      -3.234 -54.949 -45.065  1.00 20.00           C

You can open the file in any text editor (like textedit, nedit, vi, but not a word processor), delete the two extra spaces that come after the “HETATM” for those atoms, and everything should line up and be read properly. You should also delete the TER line that comes after Val 145, that indicates a chain termination.

hth

Kevin M. Jude, PhD

Structural Biology Research Specialist, Garcia Lab

Howard Hughes Medical Institute

Stanford University School of Medicine

Beckman B177, 279 Campus Drive, Stanford CA 94305

From: TheWildBynapie via ChimeraX-users <chimerax-users@cgl.ucsf.edu>
Date: Thursday, May 14, 2026 at 2:18 PM
To: chimerax-users@cgl.ucsf.edu <chimerax-users@cgl.ucsf.edu>
Subject: [chimerax-users] Covalently attaching unusual ligands

Hi there, I'm a bioinformatics UCT student and we've been given an assignment to solve a protein structure. Everyone has been struggling with this step and I've tried many many ways to get this to work in ChimeraX, but I get new errors every time so I'm asking for help. Sorry for the long question, but I would highly appreciate help at this point (and so would my class - so if I get an answer you'll be making ~30 people very happy, not just 1!)

Essentially this ligand has a sulphur that makes a thioester to Cys 146, our professor said we should remove the Cys sulphur and then combine the ligand and the remainder of the ligand into one residue and name it 'AKG.' We have a dimer so we also do this in 2 places.

We need to refine this using ISOLDE, which is where most of my problems appear. Then he says we can use 'AKG.xml' in residue parameterisation so ISOLDE will accept our residue. Currently the way my pdb is layed out causes chimera to only render the ligand part of AKG but not the Cys part. I don't know why, and more importantly my general understanding of how ChimeraX reads pdb files is lacking.

So I suppose finally my actual question: what do I need to do to make it so ChimeraX renders the residue correctly, and ISOLDE accepts AKG.xml and can actually run a simulation on this pdb?

I've attached:

1: AKG.xml (for residue parameterisation)

2: 5 - refine1.mtz (the electron density map data)

3: 6c - part of AKG missing.pdb (the current pdb I'm working with where the Cys part of residue 146 isn't rendered)

4: 6b - pre-covalent linking.pdb (the latest copy of the pdb I could get running in ISOLDE simulations. This has the protein dimer and 2 ligands as 3 separate models. I'd already deleted the Cys's Sulphur here so it's more like an Ala in this case)

Sorry for such a massive email, but I wanted to be as specific as possible with my problem. If you could provide me some help it would be extremely helpful. Sadly our lectures for this module are over and getting our lecturer to explain anything is virtually impossible.

Thanks so much,

Byron (and the rest of my class to be honest)