<div dir="ltr">Hi Jorge,<div><br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">> Curiously , for some of the bonds to be added, I receive the message:<br>> " Atom "HETATM 9835 O2 3GR N 5 .*. O " rejected from bonding due to valence issues."<br>> which seems to point to oxygen atoms, though I declare carbon atoms.</blockquote><div><br></div><div>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.</div><div><br></div><div>Best regards,</div><div>Oleg Sobolev.</div><div> </div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Fri, Jul 10, 2020 at 6:11 AM Jorge Iulek <<a href="mailto:iulek@uepg.br">iulek@uepg.br</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">
<div bgcolor="#FFFFFF">
<p> 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...).</p>
<p> 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).<br>
</p>
<div> 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.</div>
<div><br>
</div>
<div><br>
-------- Forwarded Message --------
<table cellspacing="0" cellpadding="0" border="0">
<tbody>
<tr>
<th valign="BASELINE" nowrap align="RIGHT">Subject:
</th>
<td>Re: [phenixbb] ligand possibly bound to active site
cysteine</td>
</tr>
<tr>
<th valign="BASELINE" nowrap align="RIGHT">Date: </th>
<td>Thu, 9 Jul 2020 10:21:10 -0300</td>
</tr>
<tr>
<th valign="BASELINE" nowrap align="RIGHT">From: </th>
<td>Jorge Iulek <a href="mailto:iulek@uepg.br" target="_blank"><iulek@uepg.br></a></td>
</tr>
<tr>
<th valign="BASELINE" nowrap align="RIGHT">To: </th>
<td>Roger Rowlett <a href="mailto:rrowlett@colgate.edu" target="_blank"><rrowlett@colgate.edu></a></td>
</tr>
<tr>
<th valign="BASELINE" nowrap align="RIGHT">CC: </th>
<td>PHENIX user mailing list
<a href="mailto:phenixbb@phenix-online.org" target="_blank"><phenixbb@phenix-online.org></a></td>
</tr>
</tbody>
</table>
<br>
<br>
<p>Thanks Dr. Rowlett for suggestions.</p>
<p> 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.</p>
<p>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).</p>
<p>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.<br>
</p>
<p>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.</p>
<p>Should you have any other suggestion, I would be happy to here.</p>
<p>Yours,</p>
<p><br>
</p>
<p>Jorge<br>
</p>
<div><br>
</div>
<blockquote type="cite">
<div dir="auto">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.
<div dir="auto"><br>
</div>
<div dir="auto">Roger Rowlett</div>
<div dir="auto">Gordon & Dorothy Kline Professor, Emeritus</div>
<div dir="auto">Dept of Chemistry</div>
<div dir="auto">Colgate University </div>
</div>
<br>
<div class="gmail_quote">
<div dir="ltr" class="gmail_attr">On Thu, Jul 9, 2020, 6:28 AM
Jorge Iulek <<a href="mailto:iulek@uepg.br" target="_blank">iulek@uepg.br</a>> wrote:<br>
</div>
<blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">
<div bgcolor="#FFFFFF">
<p>Dear all,</p>
<p><br>
</p>
<p> I am refining a structure of a Glyceraldehyde
3-phosphate dehydrogenase (GAPDH) (converts
glyceraldehyde 3-phosphate in<font color="#330033">to <font size="+1">D<span style="font-family:sans-serif;font-size:14px;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;background-color:rgb(144,238,144);text-decoration-style:initial;text-decoration-color:initial;float:none;display:inline">-</span></font></font><a href="https://en.wikipedia.org/wiki/Glycerate_1,3-bisphosphate" title="" style="text-decoration:underline;color:rgb(250,167,0);background:none;outline-color:rgb(51,102,204);font-family:sans-serif;font-size:14px;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:-webkit-center;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px" rel="noreferrer" target="_blank"><font size="+1" color="#330033">glycerate
1,3-bisphosphate) ,
https://www.brenda-enzymes.org/enzyme.php?ecno=1.2.1.12
.</font><br>
</a></p>
<p> It turns out that its active center cysteine
presents bound ligands , covalently or not to be
determined if possible (data resolution 2.51 A).<br>
</p>
<p> 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.</p>
<p>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.</p>
<p>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).<br>
</p>
<p>Catalytic mechanism includes a hemithioacetal
intermediate (<a href="https://febs.onlinelibrary.wiley.com/doi/abs/10.1046/j.1432-1327.1998.2520447.x" rel="noreferrer" target="_blank">
https://febs.onlinelibrary.wiley.com/doi/abs/10.1046/j.1432-1327.1998.2520447.x</a>
) such that cysteine SD is bound covalently to a carbon.
I wonder also how much this might attack an ethylene
glycol and their likes.</p>
<p>Pictures for the density are shown at for the 4
monomers of the a. u., first 4 photos: <a href="https://photos.app.goo.gl/Y7MyugqwRFD4sjgDA" rel="noreferrer" target="_blank">https://photos.app.goo.gl/Y7MyugqwRFD4sjgDA</a>
(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.</p>
<p>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).</p>
<p>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:</p>
<p> bond {<br>
action = *add delete change<br>
atom_selection_1 = chain A and resid 153 and name
SG<br>
atom_selection_2 = chain N and resid 5 and name C3<br>
symmetry_operation = None<br>
distance_ideal = 1.803<br>
sigma = 0.1<br>
slack = None<br>
limit = -1.0<br>
top_out = False<br>
}<br>
</p>
<p> Results are also show for my Glyceraldehyde trial,
last 4 photos, <a href="https://photos.google.com/album/AF1QipO71L7GJYKv_MmjTc_0GzsH2xtFR_V-2ICBirPb" rel="noreferrer" target="_blank">https://photos.google.com/album/AF1QipO71L7GJYKv_MmjTc_0GzsH2xtFR_V-2ICBirPb</a>
. Note clashes.</p>
<p> Curiously , for some of the bonds to be added, I
receive the message:<br>
</p>
<p>" Atom "HETATM 9835 O2 3GR N 5 .*. O "
rejected from bonding due to valence issues."</p>
<p> which seems to point to oxygen atoms, though I
declare carbon atoms.<br>
</p>
<p><br>
</p>
<p> Helps welcome, thank you.</p>
<p><br>
</p>
<p>Jorge<br>
</p>
</div>
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