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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; -webkit-text-stroke-width: 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"
class="mw-redirect" 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; orphans: 2; text-align: -webkit-center; text-indent:
0px; text-transform: none; white-space: normal; widows: 2;
word-spacing: 0px; -webkit-text-stroke-width: 0px;"
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
class="moz-txt-link-freetext"
href="https://febs.onlinelibrary.wiley.com/doi/abs/10.1046/j.1432-1327.1998.2520447.x"
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 class="moz-txt-link-freetext"
href="https://photos.app.goo.gl/Y7MyugqwRFD4sjgDA">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 class="moz-txt-link-freetext"
href="https://photos.google.com/album/AF1QipO71L7GJYKv_MmjTc_0GzsH2xtFR_V-2ICBirPb">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>
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