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<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">
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<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">
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<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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