[3dem] Tomography to visualize interaction of liposomes

[Schmid, Michael F.]

?Is the protein layer associated with either (or both) the layers of surface ice contamination? That would be a clue.

________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Schmid, Michael F. <m-schmid at slac.stanford.edu>
Sent: Thursday, August 10, 2017 10:04 PM
To: Sonali Dhindwal; 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Tomography to visualize interaction of liposomes


?Possibly, the protein is attracted to the air-water interface. In bulk biochemical measurements (maybe also with negative stain), this is not a problem; and with single particle methods, you can't detect the problem you are seeing in tomograms, but with the thin layer after blotting, there is a lot more surface to volume ratio for the protein to be interacting with. Just a guess.

________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Sonali Dhindwal <sdhindwal at ccny.cuny.edu>
Sent: Thursday, August 10, 2017 9:56 PM
To: 3dem at ncmir.ucsd.edu
Subject: [3dem] Tomography to visualize interaction of liposomes

?Dear 3DEM group members,

I have been studying the interaction of my protein of interest (~900 KDa and ~200Å in size) with liposomes (~2000Å is size).
Biochemical assays demonstrates their interaction and so is the images collected by negative stain and Cryo-EM.
To clearly visualize interaction, I have collected few tomograms. Though, after processing the tomograms and going through the slices, liposomes and protein seem to be in different layers.
In initial slices, I can see all the liposomes and in the later slices I see all the protein molecules. And going through the slices, first I see liposomes and as they start to fade away, I start to see the protein molecules and few protein molecules lying very close to the liposomes.
Is this normal ?
I am looking for any suggestions or advice that can be done either during processing of data or preparation of grids  that can help to improve the visualization of interactions among these molecules.
Thanks and Regards

--
Sonali Dhindwal
Post Doc

"The dream is not that you see in sleep, dream is which does not let you sleep."?

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