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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">
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      <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 --------
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              <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">&lt;iulek@uepg.br&gt;</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">&lt;rrowlett@colgate.edu&gt;</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">&lt;phenixbb@phenix-online.org&gt;</a></td>
            </tr>
          </tbody>
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        <br>
        <br>
        <meta http-equiv="Content-Type" content="text/html;
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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">
          <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 &amp; 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 &lt;<a href="mailto:iulek@uepg.br"
                moz-do-not-send="true">iulek@uepg.br</a>&gt; 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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