[3dem] Interpretation of strange objects in a cryo-EM sample

[Howard Young]

Hello all,

Is it possible it is a disordered sponge phase that formed from micelles
during freezing?

Best, Howard

On Thu, Apr 13, 2023 at 8:37 AM Mariena Silvestry Ramos <ms3289 at cornell.edu>
wrote:

> Hi James and all,
>
> I agree. I've seen "webby" stuff like that too, even in protein preps,
> which I've attributed in the past to denatured/mesbehaving protein.
> Unfortunately I don't have an x-ray detector on any of my columns to
> quickly check what's in them, but that would be a possibility in a system
> that does have it.
>
> The items in question do look like micelles, and as Vincent pointed out,
> we don't have info on what the contents/combo of lipids and detergents are.
>
> Best,
>
> Mariena
>
> Sent from my Game Boy
>
> On Apr 13, 2023, at 10:08 AM, David Michael Belnap <David.Belnap at utah.edu>
> wrote:
>
> 
>
> James,
>
>
>
> Those images look consistent with large particles that exclude water.  I
> don’t know the expected sizes of micelles, however.  If the appearance
> changes with increased exposure to the electron beam, then I would agree
> that you have beam damage.  Be careful of claims of “pure” samples.  I
> often see much more than what is claimed to be in a pure sample.  Is what
> you are looking for supposed to be smaller than these particles?  As
> Vincent suggests, you may have constructs different from what you expect.
> Your lower magnification picture suggests a diluted sample to me.
>
>
>
> David
>
>
>
>
>
> *From: *3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of vincent Chaptal <
> vincent.chaptal at ibcp.fr>
> *Date: *Thursday, April 13, 2023 at 7:58 AM
> *To: *"Kizziah, James" <kizziah4 at uab.edu>, "3dem at ncmir.ucsd.edu" <
> 3dem at ncmir.ucsd.edu>
> *Subject: *Re: [3dem] Interpretation of strange objects in a cryo-EM
> sample
>
>
>
> Dear James,
>
> you didn't say which detergent the micelles would be orinigating from, it
> changes from detergent to detergent.
>
> But in any case, beside the mix DDM/CHS which forms small micelles when
> concentrated but large objects when diluted, most micelles are much much
> smaller, say 30-50Å or so.
>
> I think there is an article by the group of Arne Moeller on detergent
> micelle observation by negative stain, I don't recall if they tried in
> cryo.
>
> Best
> Vincent
>
> Le 13/04/2023 à 15:48, Kizziah, James a écrit :
>
> Hi all,
>
>
>
> We have imaged a cryo-EM sample presumed to contain micelles and observed
> the large, white objects full of/covered with junk shown in the attached
> images. Our best guess is that these are (or were) the micelles and have
> been destroyed by the electron beam, contact with the air water interface,
> or both, after vitrification. Has anyone seen something like this before or
> does anyone have alternative interpretations?
>
>
>
> Glow-discharged lacey carbon grids plunge frozen with a Mark IV Vitrobot,
> clipped and imaged on a Glacios.
>
>
>
> Thanks,
>
> James
>
>
>
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> --
>
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>
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>
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-- 
Howard S. Young, PhD
Professor
Biochemistry, MSB 3-27
University of Alberta
Edmonton, Alberta T6G 2H7

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