[3dem] question about preferred orientation

[Snijder, J. (Joost)]

Dear Mohamed,


If you see fewer particles in ice after adding detergent to the sample, it can help to apply and blot your sample on the grid multiple times before you plunge-freeze. We described the procedure some time ago here:

https://www.sciencedirect.com/science/article/pii/S104784771730031X

I had the exact issue you described with an Epstein Barr Virus protein complex. Adding the detergent (0.01% NP40 in my case) improved the orientations for me, and multiple rounds of blotting improved the numbers of particles in ice. We have also seen it work with DDM detergent. Hope this trick also works for you, good luck!


Best,


Joost

________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Mohamed A Sayed <mohamed.sobhy at kaust.edu.sa>
Sent: Thursday, May 30, 2019 7:26:01 PM
To: 3dem at ncmir.ucsd.edu
Subject: [3dem] question about preferred orientation


If you would please advise on the following:

I have protein that shows strong preferential orientation. The buffer only contains (20 mM Tris. HCl pH7.5, 150 mM NaCl).

I tried adding detergent CHAPS (8 mM final concentration) and the number of particles dropped to only a few per hole and most of the protein particels were on the carbon film. I tried different hole size gold grids r1.2/1.3 and r2/2 but the same result. Any advice how to obtain random distribution in such case.


Is there an adjustment in Relion 3d classification parameters in order to obtain a 3d classification? I tried without symmetry and both the initial model and 3d classifcation fail.

number of classes

Initial angular sampling

symmetry

CTF correction  or  'Ignore CTF’s until first peak'.

'Initial angular sampling'.

mask​

SGD:

Final resolution

Initial mini-batch size​


Thank you very much for your help.
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