[3dem] question about preferred orientation

[Christopher Arthur]

Hi Mohamed,
I would suggest the following.
1- Look at the pI of your protein and try to adjust the pH of you buffer to
give your sample a net negative charge.
2- Screen a number of detergents at multiple concentrations, i.e. 1x CMC,
0.75x CMC, 0.5x CMC (salt concentrations can effect the CMC of your
detergent, which in turn will effect how many detergent monomers are
available to bind to the air-water interface).
The detergents that I screen include a couple of anionics, a couple of
cationics, a couple of nonionics, and a couple of zwittergents (i.e. Fl Fos
Choline, CTAB, DOC, LDAO, CHAPSO, Tween20, DDM, OG).
3- You could also look at other potential additives to mask surface charge
both on your sample, on the grid surface, and at the air-water interface.

Happy to converse more about this.

Cheers,
Chris

On Thu, May 30, 2019 at 10:26 AM Mohamed A Sayed <mohamed.sobhy at kaust.edu.sa>
wrote:

> 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.
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20190530/0b81156c/attachment-0001.html>