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

Thu Apr 13 08:50:37 PDT 2023

Hi all,

Thank you for the suggestions and comments so far. These new options and possibilities are very helpful.

At the PI’s request, I must withhold some details. The solution is a made of nano- and microaggregates. I have been shown cryo-EM images of the same solution at a different concentration (micromolar range) that do show what appear to be micelles up to ~200nm, but we don’t know how these “micelles” would be structured at that size (or the larger size in the images I provided). The difference in concentration is expected to be the reason for the difference in size and morphology.

Best regards,
James

From: Alexandre Cassago <alexandre.cassago at gmail.com>
Sent: Thursday, April 13, 2023 10:40 AM
To: Mariena Silvestry Ramos <ms3289 at cornell.edu>
Cc: David Michael Belnap <David.Belnap at utah.edu>; Kizziah, James <kizziah4 at uab.edu>; 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Interpretation of strange objects in a cryo-EM sample

Dear James,

Certainly the image you showed contains burned samples, either because the dose you are using is too high (I don't really believe that, because all the carbon that surrounds the holes in the lacey grid doesn't look burned / marked), but it could be that the thickness of the ice is too thin for the size of those particles that are close to or larger than 500 nm.

Another indication that the thickness of the ice is too thin for this sample size is the location of the particles near the edges of the holes (thinner ice in the center of the hole pushes particles closer to the edges with thicker ice).

Maybe you can try thickening the ice a bit and comparing.
Another point is the concentration that really seems low, perhaps with a greater thickness of ice more particles stay in the holes or a higher concentration would be necessary.

You can also leave a time (5 to 30s - called wait time) between applying the sample to the grid and blotting before dipping in ethane, so that the sample "settles" in the holes. For some samples this really helps, for others it doesn't matter.

Here is an article with several tips and observations for studying particles like yours. The article is from 2011, but it's still very good.

Best,
Alexandre

On Apr 13, 2023, at 7:36 AM, Mariena Silvestry Ramos <ms3289 at cornell.edu<mailto: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<mailto: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<mailto:3dem-bounces at ncmir.ucsd.edu>> on behalf of vincent Chaptal <vincent.chaptal at ibcp.fr<mailto:vincent.chaptal at ibcp.fr>>
Date: Thursday, April 13, 2023 at 7:58 AM
To: "Kizziah, James" <kizziah4 at uab.edu<mailto:kizziah4 at uab.edu>>, "3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>" <3dem at ncmir.ucsd.edu<mailto: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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