[phenixbb] ligand possibly bound to active site cysteine

[Roger Rowlett]

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> 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=1.2.1.12
> .
> <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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