[phenixbb] how to set up a distance restraint ; was: Fwd: Re: ligand possibly bound to active site cysteine
Oleg Sobolev
osobolev at lbl.gov
Fri Jul 10 11:41:03 PDT 2020
Hi Jorge,
> 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.
This could be a bug. I'd be happy to investigate what is going on if you
send me the inputs (off-list) to reproduce the behavior. This being model,
any restraint files and parameters. No data is needed.
Best regards,
Oleg Sobolev.
On Fri, Jul 10, 2020 at 6:11 AM Jorge Iulek <iulek at uepg.br> wrote:
> I was advised that crystallization mixes might be contaminated with
> aldehydes and peroxides (oxidant); I should also consider a thioester (long
> time of contact among components in the crystallization mix...).
>
> I would like to clarify that the photos were made from the same
> crystal, just 2 situations (for each of the 4 monomers in the a. u.): i)
> nothing in the density and ii) one ligand (glyceraldehyde) in the density
> (this to illustrate that I could not set it apart from the Cysteine).
> Nevertheless, I still do not know how to set up a distance restraint ,
> this is what I would need now to proceed with ligand testing.
>
>
> -------- Forwarded Message --------
> Subject: Re: [phenixbb] ligand possibly bound to active site cysteine
> Date: Thu, 9 Jul 2020 10:21:10 -0300
> From: Jorge Iulek <iulek at uepg.br> <iulek at uepg.br>
> To: Roger Rowlett <rrowlett at colgate.edu> <rrowlett at colgate.edu>
> CC: PHENIX user mailing list <phenixbb at phenix-online.org>
> <phenixbb at phenix-online.org>
>
> 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.
>
> Yours,
>
>
> Jorge
>
> 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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