[3dem] DNA-protein cross-linker

[Zannati Zaoti]

Dear Alexandre,

Thank you very much for sharing this.


Regards
Zaoti

From: Alexandre Cassago <alexandre.cassago at gmail.com>
Date: Thursday, 25 January 2024 at 2:38 am
To: Zannati Zaoti <z.zaoti at uq.edu.au>
Cc: 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>, 3dem-request at ncmir.ucsd.edu <3dem-request at ncmir.ucsd.edu>
Subject: Re: [3dem] DNA-protein cross-linker

Dear Zaoti,

A long time ago we used DSS crosslink (attached) and it worked very well. The protein complexes, depending on their isoforms, assembled long filaments with increased activities compared to the well-known tetramers, however when we reduced the concentration for NS microscopy essay the filaments disassembled. Crosslinking helped us visualize naturally occurring filaments, but at lower concentrations required by NS microscopy.

Hope it helps you.


Best,
Alexandre





On Jan 23, 2024, at 18:08, Zannati Zaoti <z.zaoti at uq.edu.au> wrote:


Dear all,

I am looking for advice from experienced individuals who have performed cross-linking of DNA-bound protein complexes for cryo-EM sample preparation.
My work involves a protein that can bind to dsDNA and form filaments; however, these filaments are flexible and tend to fall apart during grid preparation. As a result, I cannot find any filaments during the cryoEM screening.
Thereafter, I have tried using glutaraldehyde and formaldehyde as cross-linkers to stabilize the filaments, but they have made the filaments even more unstable. At present, I am experimenting with DMS, but it seems that DMS neither makes the filament unstable nor stable enough for cryo-conditions.
Any advice or comment would be highly appreciated. Thank you!


Regards
Zaoti


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