[3dem] Membrane protein dissociating from nanodisc

[Dykstra, Holly]

Tim,
We use MSP1E3. Have you embedded single TM in SMALP and seen the protein stay in intact?
-Holly


From: Timothy Dafforn <T.R.Dafforn at bham.ac.uk>
Sent: Friday, October 15, 2021 10:27 AM
To: jacopo.marino at psi.ch; Dykstra, Holly <Holly.Dykstra at vai.org>
Cc: 3dem at ncmir.ucsd.edu
Subject: [External] Re: [3dem] Membrane protein dissociating from nanodisc


what form of nano disc are you using?

We do a fair amount of work on the SMALP system (we developed it) so might be able to help

Tim




Tim R. Dafforn PhD FIET
Professor of Biotechnology
University of Birmingham
Edgbaston
Birmingham
B152TT
+44 (0) 121 414 5881
________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>> on behalf of jacopo.marino at psi.ch<mailto:jacopo.marino at psi.ch> <jacopo.marino at psi.ch<mailto:jacopo.marino at psi.ch>>
Sent: 15 October 2021 14:39:07
To: Dykstra, Holly
Cc: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Membrane protein dissociating from nanodisc

Hi - one would expect that membrane proteins embedded into nanodiscs would suffer less than detergent-embedded proteins, but that's not a rule. The question is simple: is it faster to live with your small amount of particles and compensate with collecting many more datasets, or screening for freezing conditions where you have less dissociation ? You might find that areas with thicker ice on your grid have more intact particles, but likely you don't want to collect from there.

Cheers
Jacopo

Dr. Jacopo Marino Laboratory of Biomolecular Research Paul Scherrer Institute OFLB/005 5232

Villigen PSI, Switzerland

tel: +41 56 310 5777<tel:+41%2056%20310%205777>

e-mail: jacopo.marino at psi.ch<mailto:jacopo.marino at psi.ch>

On 15 Oct 2021, at 15:13, Dykstra, Holly <Holly.Dykstra at vai.org<mailto:Holly.Dykstra at vai.org>> wrote:
Hello all,
I am working on a single-TM membrane protein in nanodisc. Upon freezing grids, I am observing protein dissociation from the nanodisc. A small percentage of the total particle population is intact protein-nanodisc complex but most of the particles have dissociated. I believe this is happening at the point of freezing as negative stain shows the intact protein-nanodisc complex. Has anyone else experienced this problem or does anyone have any suggestions or advice?
Thanks!
-Holly
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