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 To: Roger Rowlett CC: PHENIX user mailing list 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 mailto:[email protected]> 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.1432-1327.1998.252044... ) 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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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 To: Roger Rowlett CC: PHENIX user mailing list

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 mailto:[email protected]> 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.1432-1327.1998.252044... ) 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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