[3dem] protein can't go to holes of cryo EM grids

[SSS Sasha]

Hi Ryan and Rajiv,

Thanks for those suggestions. Yes, I think there are some problems with GO. I will try optimize it.

Thanks so much!
Shasha

On Oct 10, 2022, at 4:05 PM, Ryan Abdella <abdel104 at umn.edu<mailto:abdel104 at umn.edu>> wrote:

Hi Sasha,

To add to what Rajiv said, it’s not clear from your image that there is even GO coating the hole that you are imaging. If have you a lot of GO on your grid, then you should see lines in your images representing the edges of GO sheets (see attached image for an egregious example). If you don’t have much GO on your grid, an atlas of a square should indicate that holes appear different (GO covered areas are usually darker) depending on the presence/absence of GO.

Best,
Ryan

<GO_grid.png>

On Oct 10, 2022, at 4:36 PM, Rajiv Ranjan Singh <rajssai at gmail.com<mailto:rajssai at gmail.com>> wrote:

Hey Sasha,

Try H2, O2 Plasma Cleaning for 30S in each case. If you see good particles in Negative stains.

I hope you are doing plasma cleaning before coating GO, if not then try following protocol from Eva Nogales lab.

Best,
Rajiv Ranjan Singh



On Tue, Oct 11, 2022 at 4:47 AM SSS Sasha <sss_jiandan at outlook.com<mailto:sss_jiandan at outlook.com>> wrote:
Dear all,

I was trying to freeze cryo EM grids for a protein ~ 0.73MDa under several conditions, but it didn't work well. I make a brief summary here. Does anyone have any suggestions? Thanks in advance!

Holly grids CF-1.2/1.3-4Cu-50: plasma cleaning for 10s using O2,Ar2            Protein can't go in to the hole

Lacey carbon 400mesh Cu:  plasma cleaning for 15s using O2, Ar2        Contrast is quite low, hardly see the particles

GO on Quantifoil: plasma cleaning for 15s using H2, O2              Protein can't go into hole

Best regards,
Shasha
<Screen Shot 2022-10-10 at 1.31.48 PM.png>
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