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

Fri Jun 5 11:33:17 PDT 2020

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!QW1Lx4uKjFGmq4z4xqmdpZ0plzZ0et43I7FvRDeqS7br-4NAg7NpvqQekiobeXNF8Q$ ). 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
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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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