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

[Rajiv Ranjan Singh]

Hey Sasha,

If you need, I can send you my protocol revised based on Eva Nogales lab
and protocol provided by Matthew Belousoff (Monash University). This covers
almost 60-70 of the holes on UltraAufoil grids!!

Best,
Rajiv Ranjan Singh


On Tue, Oct 11, 2022 at 9:32 AM SSS Sasha <sss_jiandan at outlook.com> wrote:

> 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> 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> 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> 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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