[3dem] Hexamer dissociate while freezing Cryo-EM grids
Sacha De Carlo
sacha.decarlo at dectris.com
Fri Jun 5 22:41:06 PDT 2020
Hi All,
some useful insights - perhaps - may be in this book chapter I co-wrote
with Robin Harris, whose publications are worth checking out, as far as
negative staining is concerned.
All the best & stay safe.
Sacha
On Sat, Jun 6, 2020 at 1:06 AM Craig, Roger <Roger.Craig at umassmed.edu>
wrote:
> 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>
> 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!SX43zkedKY6zZda1WDGQuGY2JF-kfpOhRZlZ489bH6k3LNpuHm05dnBp0KO9wj5juw$
> <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
>
> ***********************************************************************
> Roger Craig Phone: (508) 856
> 2474
> Division of Cell Biology and Imaging Fax: (508) 856 1033
> Department of Radiology Roger.Craig at umassmed.edu
> Rm. S7-210
> UMass Medical School
> 55 Lake Ave. North
> Worcester, MA 01655, USA
>
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> -----Original Message-----
> From: 3dem <3dem-bounces at ncmir.ucsd.edu> On Behalf Of Reinhard Rachel
> Sent: Friday, June 5, 2020 10:28 AM
> To: farzaad at gmail.com; noonzhou at gmail.com
> Cc: 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
> 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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