[3dem] Hexamer dissociate while freezing Cryo-EM grids

Fri Jun 5 16:06:15 PDT 2020

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.

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.

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.

Best,
Roger

From: Zhongwu Zhou <noonzhou at gmail.com>
Sent: Friday, June 5, 2020 2:33 PM
To: Craig, Roger <Roger.Craig at umassmed.edu>
Cc: Reinhard Rachel <Reinhard.Rachel at biologie.uni-regensburg.de>; farzaad at gmail.com; 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Hexamer dissociate while freezing Cryo-EM grids

Hi Roger,

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?

best,

zhongwu

On Fri, Jun 5, 2020 at 10:03 AM Craig, Roger <Roger.Craig at umassmed.edu<mailto:Roger.Craig at umassmed.edu>> wrote:
I think the demonstration of UA being a "fixative" (on the millisecond timescale) comes from our 2003 JSB paper (https://urldefense.com/v3/__https://pubmed.ncbi.nlm.nih.gov/12576019/__;!!Mih3wA!XSAI0RBfpiRelOzHlJouX92j6uZ-RLNy6COudOP3igKWyWzg27wf1Z2KqOEbee0EcA$ <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$ >). 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.

All the best,
Roger

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-----Original Message-----
From: 3dem <3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>> On Behalf Of Reinhard Rachel
Sent: Friday, June 5, 2020 10:28 AM
To: farzaad at gmail.com<mailto:farzaad at gmail.com>; noonzhou at gmail.com<mailto:noonzhou at gmail.com>
Cc: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
Subject: [3dem] Hexamer dissociate while freezing Cryo-EM grids

>>> 05.06.2020 at 15:05:
> The pH of UA is ~4. The common buffer for protein sample is pH ~6-8.

this provokes an answer. This is questionable, IMO! many proteins investigated are cytosolic proteins (yes, not all, I know).
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.

> Depending on the settings of staining method, the lower pH can be buffered!?

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.
Just note that UO2 ions are less / barely soluble in the presence of carbonate
(CO3 (2-)), and Phosphate is even worse. precipitation ...

> Moreover, Uranyl acetate acts as a fixative,

it is not a chemical fixative - no, not like Formaldehyde or Glutaraldehyde.
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"?

> preserving many protein-protein
> interactions on a millisecond time scale.

I would say - arresting or reducing the protein's flexibility and functionality ... they get out of their physiological status ...

> Thus, can you give us some
> explosion of UA staining affecting the structure? the overall
> structure or secondary structure?

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.
just my 2 Euro-cents

kind regards,
reinhard

--
Prof. Dr. Reinhard Rachel
University of Regensburg
Centre for EM / Anatomy
Faculty of Biology & Preclin. Med.
Universitaetsstrasse 31
D-93053 Regensburg - Germany
tel +49 941 943 -2837, -1720
mail reinhard.rachel at biologie.uni-regensburg.de<mailto:reinhard.rachel at biologie.uni-regensburg.de>
office: VKL 3.1.29
member of the IFSM board

Next microscopy conferences:
- cancelled: EMC2020 in Kopenhagen, 23.-28.8. 2020 (European conference)
- MC2021 in Vienna (D-A-CH conference)
- IMC20 in Busan, South Korea: Sept 25-30, 2022
- next Microbiol. conferences:  VAAM March 2021 Düsseldorf

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