<div dir="ltr">Hi Roger,<div><br></div><div>Thanks for providing the info. Can you provide a little bit more info about how UA interacts with nucleic acids or proteins? Also, in your 2003 JSB paper, I noticed that some sample is negative stained and the other is positive stained. What cause the difference? Is it possible to estimate to the amount of heave mental absorbed on the sample? Furthermore, based on the intensity of sample on the positive stained images, can we relatively estimate the molecular mass of the sample if a reference provided in the same image conditions?</div><div><br></div><div>best,</div><div><br></div><div>zhongwu</div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Fri, Jun 5, 2020 at 10:03 AM Craig, Roger <<a href="mailto:Roger.Craig@umassmed.edu" target="_blank">Roger.Craig@umassmed.edu</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">I think the demonstration of UA being a "fixative" (on the millisecond timescale) comes from our 2003 JSB paper (<a href="https://urldefense.com/v3/__https://pubmed.ncbi.nlm.nih.gov/12576019/__;!!Mih3wA!QW1Lx4uKjFGmq4z4xqmdpZ0plzZ0et43I7FvRDeqS7br-4NAg7NpvqQekiobeXNF8Q$" rel="noreferrer" target="_blank">https://pubmed.ncbi.nlm.nih.gov/12576019/</a>). No, it is not a chemical crosslinker, but it stabilizes protein structure in some way, still not understood. As far as we could determine it was not simply due to a reduction in pH.<br>
<br>
All the best,<br>
Roger<br>
<br>
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-----Original Message-----<br>
From: 3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu" target="_blank">3dem-bounces@ncmir.ucsd.edu</a>> On Behalf Of Reinhard Rachel<br>
Sent: Friday, June 5, 2020 10:28 AM<br>
To: <a href="mailto:farzaad@gmail.com" target="_blank">farzaad@gmail.com</a>; <a href="mailto:noonzhou@gmail.com" target="_blank">noonzhou@gmail.com</a><br>
Cc: <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a><br>
Subject: [3dem] Hexamer dissociate while freezing Cryo-EM grids<br>
<br>
>>> 05.06.2020 at 15:05:<br>
> The pH of UA is ~4. The common buffer for protein sample is pH ~6-8. <br>
<br>
this provokes an answer. This is questionable, IMO! many proteins investigated are cytosolic proteins (yes, not all, I know). <br>
The pH inside the cytoplasm is at around 6 or 6.5, but not higher than 7 - and thus, the TRIS-buffered solution with a pH of 7.4 or higher as often used are not physiological (at all). Whether this is bad for the protein or not? I do not know. BUT, keep this in mind. <br>
<br>
> Depending on the settings of staining method, the lower pH can be buffered!?<br>
<br>
<br>
It is questionable whether a UAc solution with a buffer at pH >5 (or 6 or 7) is "stable"? I would not want to try out. - What happens with the UO2 ion (which in UAc solutions are complexed by acetate ions)? often, it precipitates.<br>
Just note that UO2 ions are less / barely soluble in the presence of carbonate<br>
(CO3 (2-)), and Phosphate is even worse. precipitation ... <br>
<br>
> Moreover, Uranyl acetate acts as a fixative,<br>
<br>
it is not a chemical fixative - no, not like Formaldehyde or Glutaraldehyde.<br>
If you use the term 'fixative', you should say what you mean. It is a kind of bringing the protein close to its iso-electric point (pH 4.5 +/-), where the protein is at its point of "lowest solubility". Is it what you mean with "fixative"?<br>
<br>
> preserving many protein-protein<br>
> interactions on a millisecond time scale. <br>
<br>
I would say - arresting or reducing the protein's flexibility and functionality ... they get out of their physiological status ... <br>
<br>
> Thus, can you give us some<br>
> explosion of UA staining affecting the structure? the overall <br>
> structure or secondary structure?<br>
<br>
by getting close to the iso-electric point, I do not predict how much the tertiary or secondary structure is altered. Is it altered?? It is just the ionic groups in the side chains, which become less charged, less polar. Is this denaturation? not necessarily. <br>
just my 2 Euro-cents <br>
<br>
kind regards,<br>
reinhard<br>
<br>
--<br>
Prof. Dr. Reinhard Rachel<br>
University of Regensburg<br>
Centre for EM / Anatomy<br>
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Universitaetsstrasse 31<br>
D-93053 Regensburg - Germany<br>
tel +49 941 943 -2837, -1720<br>
mail <a href="mailto:reinhard.rachel@biologie.uni-regensburg.de" target="_blank">reinhard.rachel@biologie.uni-regensburg.de</a><br>
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member of the IFSM board<br>
<br>
Next microscopy conferences:<br>
- cancelled: EMC2020 in Kopenhagen, 23.-28.8. 2020 (European conference)<br>
- MC2021 in Vienna (D-A-CH conference)<br>
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- next Microbiol. conferences: VAAM March 2021 Düsseldorf<br>
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