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

[Jiang,Qiu-Xing]

Dear Umar,
We had a case with an amphipathic protein in detergents that became suitable for cryo imaging only when being positioned tens of Å’s above the ChemiC films we developed (PMID: 24457027 and PMID: 30456382). The same method is suitable for modifications of graphene films, and it avoids ample biomass such as PEGs, DNA origamis, protein-binders, or other polymers, etc. It might be able to overcome the problems you encountered and help keep ice thin.
Best luck,

Qiu-Xing
CryoEM Center | Lab of Mol. Physiol. & Biophys. @ HWI


From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Ed Morris <Ed.Morris at icr.ac.uk>
Date: Thursday, June 4, 2020 at 4:37 PM
To: Umar Farook <umarfarook12 at gmail.com>, "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Hexamer dissociate while freezing Cryo-EM grids

[External Email]


​​​Dear Umar

One thing that could be worth checking is to image a range of ice thicknesses in your existing cryo grids. If you are just imaging in the thinnest ice it may be that you have excluded your hexamer and are just seeing smaller complexes. In that case the hexamers may be hiding in thicker ice.

This has happened to me in the past and I initially mistakenly concluded that large particles seen in negative stain were being replaced by subcomplexes when I went I imaged in cryo. On checking the thicker ice I realised that this was not in fact the case.

Best wishes

Ed Morris








________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Umar Farook <umarfarook12 at gmail.com>
Sent: 04 June 2020 20:24
To: 3dem at ncmir.ucsd.edu
Subject: [3dem] Hexamer dissociate while freezing Cryo-EM grids

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Dear All,

I would like to ask the community for suggestions on how to overcome the issue of hexamer dissociating while freezing Cryo-EM grids, the negative staining seems to be perfect hexamer at 0.1 mg/ml concentration. But the sample seems to fall apart at 1.5 mg/ml in Cryo freezing, please advise, thank you.

Best Regards,
Umar

The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP.

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