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<p class="MsoNormal"><span style="font-size:11.0pt;mso-fareast-language:EN-US">Dear Alexandre,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;mso-fareast-language:EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;mso-fareast-language:EN-US">Thank you very much for sharing this.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;mso-fareast-language:EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;mso-fareast-language:EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;mso-fareast-language:EN-US">Regards<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;mso-fareast-language:EN-US">Zaoti<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;mso-fareast-language:EN-US"><o:p> </o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><b><span style="color:black">From:
</span></b><span style="color:black">Alexandre Cassago <alexandre.cassago@gmail.com><br>
<b>Date: </b>Thursday, 25 January 2024 at 2:38</span><span style="font-family:"Arial",sans-serif;color:black"> </span><span style="color:black">am<br>
<b>To: </b>Zannati Zaoti <z.zaoti@uq.edu.au><br>
<b>Cc: </b>3dem@ncmir.ucsd.edu <3dem@ncmir.ucsd.edu>, 3dem-request@ncmir.ucsd.edu <3dem-request@ncmir.ucsd.edu><br>
<b>Subject: </b>Re: [3dem] DNA-protein cross-linker<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Dear Zaoti, <o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">A long time ago we used DSS crosslink (attached) and it worked very well. The protein complexes, depending on their isoforms, assembled long filaments with increased activities compared to the well-known tetramers, however when we reduced
the concentration for NS microscopy essay the filaments disassembled. Crosslinking helped us visualize naturally occurring filaments, but at lower concentrations required by NS microscopy.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Hope it helps you.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Best, <o:p></o:p></p>
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<p class="MsoNormal">Alexandre <o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">On Jan 23, 2024, at 18:08, Zannati Zaoti <z.zaoti@uq.edu.au> wrote:<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:#212121"> </span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:#212121">Dear all,</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:#212121"> </span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:#212121">I am looking for advice from experienced individuals who have performed cross-linking of DNA-bound protein complexes for cryo-EM sample preparation.</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:#212121">My work involves a protein that can bind to dsDNA and form filaments; however, these filaments are flexible and tend to fall apart during grid preparation. As a result, I cannot find any filaments
during the cryoEM screening.</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:#212121">Thereafter, I have tried using glutaraldehyde and formaldehyde as cross-linkers to stabilize the filaments, but they have made the filaments even more unstable. At present, I am experimenting
with DMS, but it seems that DMS neither makes the filament unstable nor stable enough for cryo-conditions. </span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:#212121">Any advice or comment would be highly appreciated. Thank you!</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:#212121"> </span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:#212121"> </span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:#212121">Regards</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt;color:#212121">Zaoti </span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.5pt;font-family:Helvetica">_______________________________________________<br>
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<p class="MsoNormal"><o:p> </o:p></p>
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