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<p class="MsoNormal">Dear Umar, <o:p></o:p></p>
<p class="MsoNormal">We had a case with an amphipathic protein in detergents that became suitable for cryo imaging only when being positioned tens of Å’s above the ChemiC films we developed (PMID: 24457027 and PMID: 30456382). The same method is suitable for
modifications of graphene films, and it avoids ample biomass such as PEGs, DNA origamis, protein-binders, or other polymers, etc. It might be able to overcome the problems you encountered and help keep ice thin.
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<p class="MsoNormal">Best luck,<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><span style="font-size:12.0pt;font-family:"Matura MT Script Capitals";color:black">Qiu-Xing
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Arial",sans-serif;color:black">CryoEM Center | Lab of Mol. Physiol. & Biophys. @ HWI<o:p></o:p></span></p>
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<p class="MsoNormal"><b><span style="font-size:12.0pt;color:black">From: </span></b><span style="font-size:12.0pt;color:black">3dem <3dem-bounces@ncmir.ucsd.edu> on behalf of Ed Morris <Ed.Morris@icr.ac.uk><br>
<b>Date: </b>Thursday, June 4, 2020 at 4:37 PM<br>
<b>To: </b>Umar Farook <umarfarook12@gmail.com>, "3dem@ncmir.ucsd.edu" <3dem@ncmir.ucsd.edu><br>
<b>Subject: </b>Re: [3dem] Hexamer dissociate while freezing Cryo-EM grids<o:p></o:p></span></p>
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<p class="MsoNormal"><b><span style="font-size:9.0pt;font-family:"Arial",sans-serif;color:#FA4616">[External Email]</span></b>
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<p>Dear Umar<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">One thing that could be worth checking is to image a range of ice thicknesses in your existing cryo grids. If you are just imaging in the thinnest ice it may be that you have excluded your hexamer and are just seeing smaller complexes.
In that case the hexamers may be hiding in thicker ice. <o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">This has happened to me in the past and I initially mistakenly concluded that large particles seen in negative stain were being replaced by subcomplexes when I went I imaged in cryo. On checking the thicker ice I realised that this was
not in fact the case.<o:p></o:p></p>
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<p class="MsoNormal">Best wishes<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Ed Morris<o:p></o:p></p>
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<p class="MsoNormal"><b><span style="color:black">From:</span></b><span style="color:black"> 3dem <3dem-bounces@ncmir.ucsd.edu> on behalf of Umar Farook <umarfarook12@gmail.com><br>
<b>Sent:</b> 04 June 2020 20:24<br>
<b>To:</b> 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> [3dem] Hexamer dissociate while freezing Cryo-EM grids</span><span style="color:#212121">
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<p class="MsoNormal"><span style="color:#212121"> <o:p></o:p></span></p>
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<p class="MsoNormal" style="line-height:12.0pt;background:#FFEB9C"><b><span style="font-size:10.0pt;color:#9C6500">CAUTION:</span></b><span style="font-size:10.0pt;color:black"> This email originated from outside of the ICR. Do not click links or open attachments
unless you recognize the sender's email address and know the content is safe.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="color:#212121">Dear All, <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="color:#212121"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="color:#212121">I would like to ask the community for suggestions on how to overcome the issue of hexamer dissociating while freezing Cryo-EM grids, the negative staining seems to be perfect hexamer at 0.1 mg/ml concentration.
But the sample seems to fall apart at 1.5 mg/ml in Cryo freezing, please advise, thank you.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="color:#212121"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="color:#212121">Best Regards,<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="color:#212121">Umar<o:p></o:p></span></p>
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<p class="MsoNormal"><br>
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