[phenixbb] ligand possibly bound to active site cysteine

Jorge Iulek iulek at uepg.br
Thu Jul 9 06:21:10 PDT 2020

Thanks Dr. Rowlett for suggestions.

I tried to verify different degrees of oxidation and there goes residues 
called CSX, CSD, CSU. In some of the cases, the density extends beyond 
an oxygen atom, in some cases maybe that could be modeled.

About glycols, in fact I would not expect them to have reacted, but I 
still would need to learn (I need help here!) how to keep them apart 
from clashing to the cysteine (setup a due distance).

The density near Thr, yes, a water molecule fits there, although in some 
case it is quite strong, slightly resembling a tetrahedron. On 
possibility might be a partial occupancy for a phosphate (in this case 
surrounding residues should turn their H towards it) , I think.

I received also a question about the presence of DTT or mercaptoethanol; 
no, they were not present. I recall a case I had cacodylate (not this 
case) and I saw reaction (of cysteine) with the arsenic moiety. I have 
here MES  buffer, but the density would not fit well a(n extra) sulfate 
like moiety.

Should you have any other suggestion, I would be happy to here.



> A possibility is that your Cys residue has been oxidized to 
> S-hydoxycysteine. The blob near the Thr could be potentially modeled 
> as a water molecule. We have seen S-hydoxycysteine in a cysteine 
> hydrolase before. It can happen if the enzyme is adventitiously 
> oxidized during purification, storage, or crystallization. Glycols 
> themselves would not be expected to be chemically reactive with Cys.
> Roger Rowlett
> Gordon & Dorothy Kline Professor, Emeritus
> Dept of Chemistry
> Colgate University
> On Thu, Jul 9, 2020, 6:28 AM Jorge Iulek <iulek at uepg.br 
> <mailto:iulek at uepg.br>> wrote:
>     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= .
>     <https://en.wikipedia.org/wiki/Glycerate_1,3-bisphosphate>
>         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
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