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<p>Dear all,</p>
<p><br>
</p>
<p> Mention is due that I received much help from Dr. Sobolev ,
who directed me to the paper <a
href="http://scripts.iucr.org/cgi-bin/paper?S0907444911047834">http://scripts.iucr.org/cgi-bin/paper?S0907444911047834</a>
that clarifies many points I cite below concerning how to set
properly custom distances and other geometrical restraints. <br>
</p>
<p> Thanks,</p>
<p><br>
</p>
<p>Jorge</p>
<p><br>
</p>
<div class="moz-cite-prefix">On 7/10/20 10:06 AM, Jorge Iulek wrote:<br>
</div>
<blockquote type="cite"
cite="mid:29c61f4e-324a-c4f2-bde3-8d7ab27de93c@uepg.br">
<meta http-equiv="content-type" content="text/html; charset=UTF-8">
<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 class="moz-forward-container"> 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 class="moz-forward-container"><br>
</div>
<div class="moz-forward-container"><br>
-------- Forwarded Message --------
<table class="moz-email-headers-table" cellspacing="0"
cellpadding="0" border="0">
<tbody>
<tr>
<th valign="BASELINE" nowrap="nowrap" align="RIGHT">Subject:
</th>
<td>Re: [phenixbb] ligand possibly bound to active site
cysteine</td>
</tr>
<tr>
<th valign="BASELINE" nowrap="nowrap" align="RIGHT">Date:
</th>
<td>Thu, 9 Jul 2020 10:21:10 -0300</td>
</tr>
<tr>
<th valign="BASELINE" nowrap="nowrap" align="RIGHT">From:
</th>
<td>Jorge Iulek <a class="moz-txt-link-rfc2396E"
href="mailto:iulek@uepg.br" moz-do-not-send="true"><iulek@uepg.br></a></td>
</tr>
<tr>
<th valign="BASELINE" nowrap="nowrap" align="RIGHT">To: </th>
<td>Roger Rowlett <a class="moz-txt-link-rfc2396E"
href="mailto:rrowlett@colgate.edu"
moz-do-not-send="true"><rrowlett@colgate.edu></a></td>
</tr>
<tr>
<th valign="BASELINE" nowrap="nowrap" align="RIGHT">CC: </th>
<td>PHENIX user mailing list <a
class="moz-txt-link-rfc2396E"
href="mailto:phenixbb@phenix-online.org"
moz-do-not-send="true"><phenixbb@phenix-online.org></a></td>
</tr>
</tbody>
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<br>
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<meta http-equiv="Content-Type" content="text/html;
charset=UTF-8">
<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 class="moz-cite-prefix"><br>
</div>
<blockquote type="cite"
cite="mid:CA+Ey6U570ffXp4HGQqHRQ4wPEnr-WYD1QSAQPgTrktNm4qNXWg@mail.gmail.com">
<meta http-equiv="content-type" content="text/html;
charset=UTF-8">
<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"
moz-do-not-send="true">iulek@uepg.br</a>> wrote:<br>
</div>
<blockquote class="gmail_quote" style="margin:0 0 0
.8ex;border-left:1px #ccc solid;padding-left:1ex">
<div text="#000000" 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;display:inline!important;float:none">-</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"
target="_blank" rel="noreferrer"
moz-do-not-send="true"><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"
target="_blank" rel="noreferrer"
moz-do-not-send="true">
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"
target="_blank" rel="noreferrer"
moz-do-not-send="true">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"
target="_blank" rel="noreferrer"
moz-do-not-send="true">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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