[phenixbb] how to set up a distance restraint ; was: Fwd: Re: ligand possibly bound to active site cysteine
Jorge Iulek
iulek at uepg.br
Sat Jul 18 08:41:43 PDT 2020
Dear all,
Mention is due that I received much help from Dr. Sobolev , who
directed me to the paper
http://scripts.iucr.org/cgi-bin/paper?S0907444911047834 that clarifies
many points I cite below concerning how to set properly custom distances
and other geometrical restraints.
Thanks,
Jorge
On 7/10/20 10:06 AM, Jorge Iulek 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>
> To: Roger Rowlett <rrowlett at colgate.edu>
> CC: PHENIX user mailing list <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
>> <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=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
>>
>> _______________________________________________
>> phenixbb mailing list
>> phenixbb at phenix-online.org <mailto:phenixbb at phenix-online.org>
>> http://phenix-online.org/mailman/listinfo/phenixbb
>> Unsubscribe: phenixbb-leave at phenix-online.org
>> <mailto:phenixbb-leave at phenix-online.org>
>>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://phenix-online.org/pipermail/phenixbb/attachments/20200718/5f49f515/attachment.htm>
More information about the phenixbb
mailing list