<html xmlns:v="urn:schemas-microsoft-com:vml" xmlns:o="urn:schemas-microsoft-com:office:office" xmlns:w="urn:schemas-microsoft-com:office:word" xmlns:m="http://schemas.microsoft.com/office/2004/12/omml" xmlns="http://www.w3.org/TR/REC-html40">
<head>
<meta http-equiv="Content-Type" content="text/html; charset=utf-8">
<meta name="Generator" content="Microsoft Word 15 (filtered medium)">
<style><!--
/* Font Definitions */
@font-face
{font-family:Calibri;
panose-1:2 15 5 2 2 2 4 3 2 4;}
/* Style Definitions */
p.MsoNormal, li.MsoNormal, div.MsoNormal
{margin:0in;
margin-bottom:.0001pt;
font-size:12.0pt;
font-family:"Times New Roman",serif;}
a:link, span.MsoHyperlink
{mso-style-priority:99;
color:blue;
text-decoration:underline;}
a:visited, span.MsoHyperlinkFollowed
{mso-style-priority:99;
color:purple;
text-decoration:underline;}
p.msonormal0, li.msonormal0, div.msonormal0
{mso-style-name:msonormal;
mso-margin-top-alt:auto;
margin-right:0in;
mso-margin-bottom-alt:auto;
margin-left:0in;
font-size:12.0pt;
font-family:"Times New Roman",serif;}
span.EmailStyle18
{mso-style-type:personal;
font-family:"Arial",sans-serif;
color:#1F497D;}
span.EmailStyle19
{mso-style-type:personal-compose;
font-family:"Arial",sans-serif;
color:windowtext;}
.MsoChpDefault
{mso-style-type:export-only;
font-size:10.0pt;
font-family:"Calibri",sans-serif;}
@page WordSection1
{size:8.5in 11.0in;
margin:1.0in 1.0in 1.0in 1.0in;}
div.WordSection1
{page:WordSection1;}
--></style><!--[if gte mso 9]><xml>
<o:shapedefaults v:ext="edit" spidmax="1026" />
</xml><![endif]--><!--[if gte mso 9]><xml>
<o:shapelayout v:ext="edit">
<o:idmap v:ext="edit" data="1" />
</o:shapelayout></xml><![endif]-->
</head>
<body lang="EN-US" link="blue" vlink="purple">
<div class="WordSection1">
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif;color:#1F497D">Hi Zhongwu – as far as I know, not a lot is known about how the stain interacts with nucleic acids and proteins. As I understand it, the bottom line is that it
uniformly coats the surface of the protein (uranyl ions binding to negatively charged residues?), sometimes with specific patches of positive staining, but usually not penetrating the protein. When the stain dries, the protein looks pale due to exclusion of
stain compared to the surrounding puddle in which it lies. I don’t think UA works well with nucleic acids. Not sure why.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif;color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif;color:#1F497D">I think most of our JSB images showed true negative staining. When there is positive staining, it is usually due to hydrophobic properties of the carbon, so the
stain does not spread out around the protein. And there can be local positively stained features due to specific binding mentioned above.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif;color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif;color:#1F497D">I don’t know whether it’s possible to estimate how much stain is bound. I guess it could be done, but can’t think of any paper where this has been tried. It’s an
interesting idea to try to estimate relative molecular mass in this way. I haven’t heard of this being tried either. Mostly people use STEM measurement of unstained particles.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif;color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif;color:#1F497D">Best,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif;color:#1F497D">Roger<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif;color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><b><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">From:</span></b><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"> Zhongwu Zhou <noonzhou@gmail.com>
<br>
<b>Sent:</b> Friday, June 5, 2020 2:33 PM<br>
<b>To:</b> Craig, Roger <Roger.Craig@umassmed.edu><br>
<b>Cc:</b> Reinhard Rachel <Reinhard.Rachel@biologie.uni-regensburg.de>; farzaad@gmail.com; 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> Re: [3dem] Hexamer dissociate while freezing Cryo-EM grids<o:p></o:p></span></p>
<p class="MsoNormal"><o:p> </o:p></p>
<div>
<p class="MsoNormal">Hi Roger,<o:p></o:p></p>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">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?<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">best,<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">zhongwu<o:p></o:p></p>
</div>
</div>
<p class="MsoNormal"><o:p> </o:p></p>
<div>
<div>
<p class="MsoNormal">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:<o:p></o:p></p>
</div>
<blockquote style="border:none;border-left:solid #CCCCCC 1.0pt;padding:0in 0in 0in 6.0pt;margin-left:4.8pt;margin-top:5.0pt;margin-right:0in;margin-bottom:5.0pt">
<p class="MsoNormal">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://nam01.safelinks.protection.outlook.com/?url=https*3A*2F*2Fpubmed.ncbi.nlm.nih.gov*2F12576019*2F&data=02*7C01*7CRoger.Craig*40umassmed.edu*7C37a95e88dff34322be9208d8097f004f*7Cee9155fe2da34378a6c44405faf57b2e*7C0*7C0*7C637269788371446264&sdata=q*2Bw2qJ*2FrJ*2BgZ05JblKoMNeLLf3fnuN*2FI4YYwW0K2Z0Y*3D&reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUl!!Mih3wA!XSAI0RBfpiRelOzHlJouX92j6uZ-RLNy6COudOP3igKWyWzg27wf1Z2KqOHNZmavrQ$" 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>
***********************************************************************<br>
Roger Craig Phone: (508) 856 2474<br>
Division of Cell Biology and Imaging Fax: (508) 856 1033<br>
Department of Radiology <a href="mailto:Roger.Craig@umassmed.edu" target="_blank">
Roger.Craig@umassmed.edu</a><br>
Rm. S7-210 <br>
UMass Medical School <br>
55 Lake Ave. North <br>
Worcester, MA 01655, USA <br>
<br>
Confidentiality Notice:<br>
This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential, proprietary and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the
intended recipient, please contact the sender immediately and destroy or permanently delete all copies of the original message.<br>
<br>
<br>
<br>
<br>
-----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>
Faculty of Biology & Preclin. Med.<br>
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>
office: VKL 3.1.29<br>
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>
- IMC20 in Busan, South Korea: Sept 25-30, 2022<br>
- next Microbiol. conferences: VAAM March 2021 Düsseldorf<br>
<br>
_______________________________________________<br>
3dem mailing list<br>
<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a><br>
<a href="https://urldefense.com/v3/__https://nam01.safelinks.protection.outlook.com/?url=https*3A*2F*2Fmail.ncmir.ucsd.edu*2Fmailman*2Flistinfo*2F3dem&data=02*7C01*7CRoger.Craig*40umassmed.edu*7C37a95e88dff34322be9208d8097f004f*7Cee9155fe2da34378a6c44405faf57b2e*7C0*7C0*7C637269788371456253&sdata=5*2BoDi25Arxg*2FuJK8g2Ww0SQhz7tdVX3X0Z1Fk1Edh5U*3D&reserved=0__;JSUlJSUlJSUlJSUlJSUlJSU!!Mih3wA!XSAI0RBfpiRelOzHlJouX92j6uZ-RLNy6COudOP3igKWyWzg27wf1Z2KqOHSrBZ5xw$" target="_blank">https://nam01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fmail.ncmir.ucsd.edu%2Fmailman%2Flistinfo%2F3dem&data=02%7C01%7Croger.craig%40umassmed.edu%7C75187ed2df62404025b508d8095cb648%7Cee9155fe2da34378a6c44405faf57b2e%7C0%7C0%7C637269641098709226&sdata=ME%2FB%2FydDR4Tdvr5qDLVfoi9haDt2eGiQ00opDzTuxzA%3D&reserved=0</a><o:p></o:p></p>
</blockquote>
</div>
</div>
</body>
</html>