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<font face="Arial" class="">Hi Umar,</font>
<div class=""><font face="Arial" class=""><br class="">
</font></div>
<div class=""><font face="Arial" class="">You will see from the many responses that you are not alone in experiencing this problem of a specimen looking significantly different in negative stain vs cryoEM! Unfortunately, at present there is no real ‘silver
bullet’ to tackle this, and the only way forward is to test a whole load of different variables until you find the thing that works for your protein. There have been some excellent suggestions so far, but wanted to expand on these;</font></div>
<div class=""><font face="Arial" class=""><br class="">
</font></div>
<div class=""><font face="Arial" class=""><i class=""> (Caveat- only start doing these if you are confident in the quality of your prep-, you say neg stain looks nice and I expect you have biochemistry to back up (SEC (MALLS), native gels, native MS, CD, functional
assays etc).</i></font></div>
<div class=""><font face="Arial" class=""><br class="">
</font></div>
<div class=""><font face="Arial" class="">1) The first thing to check is that your particle is definitely dissociating and that you don't have intact particles. There could be other reasons why you aren’t seeing particles in the holes of your grids. The particles
could strongly partition to the carbon support film, in these cases it can sometimes be hard to distinguish particles from the amorphous carbon. If you particles are there, but on the carbon, try altering glow discharge/plasma treatment, change to a different
support material (like ultrAufoils), or investigate the use of a continuous film (GO or continuous carbon, depending on the size of your particle). As Ed pointed out, a second reason could be that your ice is so thin that you are physically excluding the particles
from the holes, in which are looking for thicker ice will confirm this, but then smaller hole diameters may help to achieve more uniform thickness ice without the thinning in the middle that leads to particle exclusion from the holes. This is most common with
larger specimens such as viruses. </font></div>
<div class=""><font face="Arial" class=""><br class="">
</font></div>
<div class=""><font face="Arial" class="">2) If you are sure the particle is dissociating, as others have pointed out the AWI is potentially to blame. There is a lot of excellent literature on why this is the case (including the excellent paper Davide pointed
out) but heres a quick summary of things you can try, in no particular order;</font></div>
<div class=""><font face="Arial" class=""><br class="">
</font></div>
<div class=""><font face="Arial" class=""><span class="Apple-tab-span" style="white-space:pre"></span>A) Move the particle away from the AWI by sequestering it at the support water interface. Includes easy-to-try options like continuous carbon (if you have
a particle where you have sufficient contrast to still align with the additional noise from the carbon), graphene oxide (or other engineered graphene supports- <a href="https://urldefense.com/v3/__https://www.pnas.org/content/116/24/11718__;!!Mih3wA!RNVnRe-vD0eh3acekU8RM5IA_xfMhvKD5diRcrMTruE-MteqcC9dzU-ZfxHSc3BrSg$" class="">https://www.pnas.org/content/116/24/11718</a>),
or ‘affinity grids’ including the srreptavadin monolayers that were mentioned, although these might involve some additional work with your protein.</font></div>
<div class=""><font face="Arial" class=""><br class="">
</font></div>
<div class=""><font face="Arial" class=""><span class="Apple-tab-span" style="white-space:pre"></span>B) Change the properties of the AWI by using a surfactant- unfortunately a huge range exist and it seems that one detergent or concentration isn’t the magic
formula that works for all proteins, so there may be a lot of variables for you to try here. Common ones to try are DDM, LMNG, CHAPSO, NP40)(stick to sub CMC concentrations for detergents). </font></div>
<div class=""><font face="Arial" class=""><br class="">
</font></div>
<div class=""><font face="Arial" class=""><span class="Apple-tab-span" style="white-space:pre"></span>C) Try a different device. The shear forces induced by filter paper based blotting might be your culprit (<a class="article-header__doi__value" href="https://urldefense.com/v3/__https://doi.org/10.1016/j.bpj.2019.12.017__;!!Mih3wA!RNVnRe-vD0eh3acekU8RM5IA_xfMhvKD5diRcrMTruE-MteqcC9dzU-ZfxFz03UNHQ$" style="box-sizing: border-box; -webkit-text-decoration-skip: objects; cursor: pointer; text-decoration: none; border-bottom-width: 2px; border-bottom-style: solid; border-bottom-color: rgba(255, 255, 255, 0.2);">https://doi.org/10.1016/j.bpj.2019.12.017</a>).
Spotiton/Chameleon/Back-It-Up/Shake-it-off, CryoWriter and time resolved cryoEM devices all avoid using filter paper. But in terms of interactions with the AWI , these occur within milliseconds- so even with the fastest of these you wont out run AWI interactions
and the potential effects (see the preprint in D)</font></div>
<div class=""><font face="Arial" class=""><br class="">
</font></div>
<div class=""><font face="Arial" class=""><span class="Apple-tab-span" style="white-space:pre"></span>D) If you can, apply a higher concentration. If the protein itself is denaturing at the AWI, it will act as a surfactant which may prevent further denaturation.
Saturating the AWI may also have positive effects on the angular distribution of particles that remain in the bulk of the thin film (away from the AWI)- <span style="color: rgb(51, 51, 51); background-color: rgb(255, 255, 255);" class=""><a href="https://urldefense.com/v3/__https://doi.org/10.1101/2020.05.14.095372__;!!Mih3wA!RNVnRe-vD0eh3acekU8RM5IA_xfMhvKD5diRcrMTruE-MteqcC9dzU-ZfxHibkKL_g$" class="">https://doi.org/10.1101/2020.05.14.095372</a>. </span></font></div>
<div class=""><font face="Arial" class=""><span style="color: rgb(51, 51, 51); background-color: rgb(255, 255, 255);" class=""><br class="">
</span></font></div>
<div class=""><font face="Arial" class=""><span style="background-color: rgb(255, 255, 255);" class=""><span class="Apple-tab-span" style="color: rgb(51, 51, 51); white-space: pre;"></span><font color="#333333" class="">E) Approach the problem from a biochemical
perspective and crosslink the sample.</font></span></font></div>
<div class=""><font face="Arial" class=""><br class="">
</font></div>
<div class=""><font face="Arial" class="">We also published a bit of a review about different variables that can be changed to alter particle distribution- <a href="https://urldefense.com/v3/__https://journals.iucr.org/d/issues/2018/06/00/ic5104/index.html__;!!Mih3wA!RNVnRe-vD0eh3acekU8RM5IA_xfMhvKD5diRcrMTruE-MteqcC9dzU-ZfxHoNQuhLQ$" class="">https://journals.iucr.org/d/issues/2018/06/00/ic5104/index.html</a></font></div>
<div class=""><font face="Arial" class=""><br class="">
</font></div>
<div class=""><font face="Arial" class="">Sorry this is a bit of a long email, but hope it is useful. Good luck! </font></div>
<div class=""><font face="Arial" class=""><br class="">
</font></div>
<div class=""><font face="Arial" class="">Best wishes,</font></div>
<div class=""><font face="Arial" class="">Becky</font></div>
<div class=""><br class="">
</div>
<div class=""><br class="">
<div class="">
<div style="color: rgb(0, 0, 0); letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">
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________________________________________________</div>
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Rebecca Thompson<br class="">
Senior cryo-Electron Microscopy Support Scientist/Facility manager <br class="">
Astbury Biostructure Laboratory<br class="">
University of Leeds<br class="">
<br class="">
Email<span class="Apple-tab-span" style="white-space: pre;"> </span><a href="mailto:r.f.thompson@leeds.ac.uk" class="">r.f.thompson@leeds.ac.uk</a><br class="">
Phone<span class="Apple-tab-span" style="white-space: pre;"> </span>0113 3433384/3438957 (office/Titan Krios control room)</div>
<div style="color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px;">
Mobile<span class="Apple-tab-span" style="white-space: pre;"> </span>07816179813<br class="">
Location<span class="Apple-tab-span" style="white-space: pre;"> </span>Roger Stevens 5.40<br class="">
Linkedin<span class="Apple-tab-span" style="white-space: pre;"> </span> <span class="Apple-tab-span" style="white-space: pre;">
</span><a href="https://urldefense.com/v3/__https://uk.linkedin.com/in/1rebeccathompson__;!!Mih3wA!RNVnRe-vD0eh3acekU8RM5IA_xfMhvKD5diRcrMTruE-MteqcC9dzU-ZfxE7B64A9w$" class="">https://uk.linkedin.com/in/1rebeccathompson</a><br class="">
Twitter<span class="Apple-tab-span" style="white-space: pre;"> </span> Bex_16</div>
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<br class="">
<div>
<blockquote type="cite" class="">
<div class="">On 5 Jun 2020, at 09:39, Umar Farook <<a href="mailto:umarfarook12@gmail.com" class="">umarfarook12@gmail.com</a>> wrote:</div>
<br class="Apple-interchange-newline">
<div class="">
<div dir="auto" class="">Thanks everyone for the quick response.
<div dir="auto" class=""><br class="">
</div>
<div dir="auto" class="">Best regards,</div>
<div dir="auto" class="">Umar</div>
</div>
<br class="">
<div class="gmail_quote">
<div dir="ltr" class="gmail_attr">On Fri, 5 Jun 2020, 5:09 am Jay Rai, <<a href="mailto:jrai@fsu.edu" class="">jrai@fsu.edu</a>> wrote:<br class="">
</div>
<blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">
<div class="">
<div dir="ltr" class="">
<div class=""></div>
<div class="">
<div class="">If your proteins complexes are too small and cannot get the enough contrast with Graphene oxide or in thicker ice then you can give a shot with strepavidine Monolayer.</div>
<div dir="ltr" class=""><br class="">
</div>
<div class=""><br class="">
</div>
<div id="m_7323023622122179323ms-outlook-mobile-signature" class="">
<div style="direction:ltr" class="">With kind regards,</div>
<div style="direction:ltr" class="">Jay</div>
</div>
</div>
</div>
<hr style="display:inline-block;width:98%" class="">
<div id="m_7323023622122179323divRplyFwdMsg" dir="ltr" class=""><font face="Calibri, sans-serif" style="font-size:11pt" class=""><b class="">From:</b> 3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu" target="_blank" rel="noreferrer" class="">3dem-bounces@ncmir.ucsd.edu</a>>
on behalf of Felipe Merino <<a href="mailto:felipe.merino@tuebingen.mpg.de" target="_blank" rel="noreferrer" class="">felipe.merino@tuebingen.mpg.de</a>><br class="">
<b class="">Sent:</b> Thursday, June 4, 2020 4:12:28 PM<br class="">
<b class="">To:</b> <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank" rel="noreferrer" class="">
3dem@ncmir.ucsd.edu</a> <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank" rel="noreferrer" class="">3dem@ncmir.ucsd.edu</a>><br class="">
<b class="">Subject:</b> Re: [3dem] [ext] Hexamer dissociate while freezing Cryo-EM grids</font>
<div class=""> </div>
</div>
<div class="">
<p class="">Hi Umar,</p>
<p class="">Or if you know already that your protein is happy on a surface you might want to give graphene or graphene oxide grids a try.
<br class="">
</p>
<p class="">Best,</p>
<p class="">Felipe<br class="">
</p>
<div class="">On 04.06.20 21:57, Schacherl, Magdalena wrote:<br class="">
</div>
<blockquote type="cite" class="">
<div class="">
<p class=""><span class="">Hi Umar,</span></p>
<div class=""><span class=""> </span><br class="webkit-block-placeholder">
</div>
<p class=""><span lang="EN-US" class="">I agree with Jay, that using detergents or surfactants (octyl glucoside 0.1% final conc. or fluorinated octyl maltoside 0.0125% final conc.) might help you. The detergents repel the protein from the water-air interface.
Be aware of the fact, that they make the ice thicker and you need a much higher concentration of protein as compared to blotting without detergents.
</span></p>
<div class=""><span lang="EN-US" class=""> </span><br class="webkit-block-placeholder">
</div>
<p class=""><span lang="EN-US" class="">If blotting per se (the force applied by the contact with filter paper) is the problem, you could try to fix the hexamer with mild glutaraldehyde treatment prior to blotting. We occasionally use
</span><span lang="EN-US" class="">0.5% (v/v) glutaraldehyde (EM grade) and incubate for 10 min at 4 °C, then immediately blot and plunge freeze.</span></p>
<div class=""><span lang="EN-US" class=""> </span><br class="webkit-block-placeholder">
</div>
<p class=""><span lang="EN-US" class="">If everything fails, then you might want to try blot-free techniques to apply your protein to grids (e.g. spotting or writing) – there are commercial machines around like Chameleon or VitroJet, or you try to spray your
protein (see S. Muench lab or J. Frank lab, and others).</span></p>
<div class=""><span lang="EN-US" class=""> </span><br class="webkit-block-placeholder">
</div>
<p class=""><span lang="EN-US" class="">Best regards, </span></p>
<div class=""><span lang="EN-US" class=""> </span><br class="webkit-block-placeholder">
</div>
<p class=""><span lang="EN-US" class="">Magdalena</span></p>
<div class=""><span lang="EN-US" class=""> </span><br class="webkit-block-placeholder">
</div>
<div class=""> <br class="webkit-block-placeholder">
</div>
<p class=""><span lang="EN-US" style="" class="">------------------------------------------------------------------------------------------------</span></p>
<p class=""><b class=""><span lang="EN-US" style="" class="">Magdalena Schacherl, PhD</span></b></p>
<p class=""><b class=""><i class=""><span lang="EN-US" style="" class="">Group Leader Structural Enzymology</span></i></b><i class=""><span lang="EN-US" style="" class=""></span></i></p>
<p class=""><span lang="EN-US" style="" class="">Charité - Universitätsmedizin Berlin</span></p>
<p class=""><span lang="EN" style="" class="">Institute of Medical Physics and Biophysics
</span><span lang="EN-US" style="" class=""> | Charitéplatz 1 | 10117 Berlin</span></p>
<p class=""><span lang="EN-US" style="" class="">Internal address: Virchowweg 6</span></p>
<p class=""><span lang="EN-US" style="" class="">Room 03.322, level 3</span></p>
<p class=""><span style="" class="">T: +49 30 450 524196 | F: +49 30 450 524952</span></p>
<p class=""><a href="mailto:magdalena.schacherl@charite.de" target="_blank" rel="noreferrer" class=""><span style="" class="">magdalena.schacherl@charite.de</span></a><span style="" class=""></span></p>
<div class=""><span style="" class=""> </span><br class="webkit-block-placeholder">
</div>
<p class=""><a href="https://urldefense.com/v3/__https://biophysik.charite.de/en/research/structural_enzymology_schacherl_lab/__;!!Mih3wA!RCBOFKYqP30RiDg5ED5tufy2vsc_-ByfjO5mvQAv7w-7fIIqLsbhR2y2YIoqQEZRjQ$" target="_blank" rel="noreferrer" class=""><span style="color:#4472c4" class="">Website
Schacherl lab</span></a><span style="color:#4472c4" class=""></span></p>
<div class=""> <br class="webkit-block-placeholder">
</div>
<div class=""><span lang="EN-US" class=""> </span><br class="webkit-block-placeholder">
</div>
<div class=""><span lang="EN-US" class=""> </span><br class="webkit-block-placeholder">
</div>
<div class=""><span lang="EN-US" class=""> </span><br class="webkit-block-placeholder">
</div>
<div style="border:none;border-top:solid #b5c4df 1.0pt;padding:3.0pt 0cm 0cm 0cm" class="">
<p class=""><b class=""><span style="font-size: 12pt;" class="">Von: </span></b><span style="font-size: 12pt;" class="">3dem
<a href="mailto:3dem-bounces@ncmir.ucsd.edu" target="_blank" rel="noreferrer" class="">
<3dem-bounces@ncmir.ucsd.edu></a> im Auftrag von Umar Farook <a href="mailto:umarfarook12@gmail.com" target="_blank" rel="noreferrer" class="">
<umarfarook12@gmail.com></a><br class="">
<b class="">Datum: </b>Donnerstag, 4. Juni 2020 um 21:25<br class="">
<b class="">An: </b><a href="mailto:3dem@ncmir.ucsd.edu" target="_blank" rel="noreferrer" class="">"3dem@ncmir.ucsd.edu"</a>
<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank" rel="noreferrer" class=""><3dem@ncmir.ucsd.edu></a><br class="">
<b class="">Betreff: </b>[ext] [3dem] Hexamer dissociate while freezing Cryo-EM grids</span></p>
</div>
<div class="">
<div class=""> <br class="webkit-block-placeholder">
</div>
</div>
<div class="">
<p class="">Dear All, </p>
<div class="">
<div class=""> <br class="webkit-block-placeholder">
</div>
</div>
<div class="">
<p class="">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.</p>
</div>
<div class="">
<div class=""> <br class="webkit-block-placeholder">
</div>
</div>
<div class="">
<p class="">Best Regards,</p>
</div>
<div class="">
<p class="">Umar</p>
</div>
</div>
</div>
<br class="">
<fieldset class=""></fieldset>
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<pre cols="72" class="">--
Felipe Merino,
Max Planck Institute for Developmental Biology
Department of Protein Evolution
Max-Planck-Ring 5
72076 Tuebingen
Phone: +49 7071 601 1351
ORCID: <a href="https://urldefense.com/v3/__https://orcid.org/0000-0003-4166-8747__;!!Mih3wA!Wovm-gVQiGlBAGs2Q9dEbwbScSTOZHyMZnEfw6hf5aactNdJuD44gYtwQi4DHBc_cA$" target="_blank" rel="noreferrer" class="">https://orcid.org/0000-0003-4166-8747</a>
Publons: <a href="https://urldefense.com/v3/__https://publons.com/a/1305699/__;!!Mih3wA!Wovm-gVQiGlBAGs2Q9dEbwbScSTOZHyMZnEfw6hf5aactNdJuD44gYtwQi75IBpG2Q$" target="_blank" rel="noreferrer" class="">https://publons.com/a/1305699/</a>
Researchgate: <a href="https://urldefense.com/v3/__https://www.researchgate.net/profile/Felipe_Merino__;!!Mih3wA!Wovm-gVQiGlBAGs2Q9dEbwbScSTOZHyMZnEfw6hf5aactNdJuD44gYtwQi7Xv37Ung$" target="_blank" rel="noreferrer" class="">https://www.researchgate.net/profile/Felipe_Merino</a></pre>
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