[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.



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= .
>>     <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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