Dear all,

    I am refining a structure of a Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (converts glyceraldehyde 3-phosphate into D-glycerate 1,3-bisphosphate) , https://www.brenda-enzymes.org/enzyme.php?ecno=1.2.1.12 .

    It turns out that its active center cysteine presents bound ligands , covalently or not to be determined if possible (data resolution 2.51 A).

    I would like to get help on two issues, (1) what the ligand might be and (2) how to treat it (correct me) in phenix.refine.

1) The protein was expressed in E. coli; it had much contact with glycerol and crystallization conditions include the "ethylene-glycols-mix" ("a mixture of diethylene glycol, triethylene glycol, tetraethylene glycol, and pentaethylene glycol"). Nevertheless, no NAD cofactor was added, and there is no electron density for it. Otherwise, phosphate was also present in crystallization condition.

In a previous study, I learned that glycerol might also contain minor amounts of ethylene glycol. I wonder, nevertheless, about glyceraldehyde (and note resemblance with the substrate).

Catalytic mechanism includes a hemithioacetal intermediate ( https://febs.onlinelibrary.wiley.com/doi/abs/10.1046/j.1432-1327.1998.2520447.x ) such that cysteine SD is bound covalently to a carbon. I wonder also how much this might attack an ethylene glycol and their likes.

Pictures for the density are shown at for the 4 monomers of the a. u., first 4 photos: https://photos.app.goo.gl/Y7MyugqwRFD4sjgDA  (blue 1 sig for e. d. maps, green 3 sig for Fourier difference maps) . Density is  different among them to different degrees. The nearby threonine, in some cases, seems to interact with a blob (and it is helped by other threonine and a serine) which + - might accommodate a phosphate.

I have tried to fit a number of molecules, e.g., the substrate (but not really good in all monomers for the phosphate moiety), glycerol, ethylene glycol and its di and tri (found also in other places in the structure) and now I went for  glyceraldehyde (though, I have doubts that there is other - apart from the one eventually bound to S - tertiary carbon). Apart from the difficulties on searching for the best fitting molecule (and consider their intrinsic flexibility) I do not manage to establish distance between them and Cys SD (and there goes the second question).

2) I could not devise how to set a proper distance between any of the ligands and the Cysteine, be it to check for a covalent bond or to establish a van der Waals restriction. I tried:

    bond {
      action = *add delete change
      atom_selection_1 = chain A and resid 153 and name SG
      atom_selection_2 = chain N and resid 5 and name C3
      symmetry_operation = None
      distance_ideal = 1.803
      sigma = 0.1
      slack = None
      limit = -1.0
      top_out = False
    }

    Results are also show for my Glyceraldehyde trial, last 4 photos,  https://photos.google.com/album/AF1QipO71L7GJYKv_MmjTc_0GzsH2xtFR_V-2ICBirPb . Note clashes.

    Curiously , for some of the bonds to be added, I receive the message:

"  Atom "HETATM 9835  O2  3GR N   5 .*.     O  " rejected from bonding due to valence issues."

    which seems to point to oxygen atoms, though I declare carbon atoms.

    Helps welcome, thank you.

Jorge