[3dem] Post-processing of dataset from carbon-coated grids: noise pb

[Francesca GUBELLINI]

Dear all,

thank you for you inputs, we wanted to know if there is a special way /trick for carbon-coated grids, since we are not used to work on these grids.

To answer some of your questions:

- We are working on a large complex (600kDa, about 250A) which is very flexible 

- we used commercial-carbon coated Qfoil R1.2/1.3, so carbon is 2nm

- Processing was done using Cryosparc and a large box size extraction (980px Fourier-cropped to 320px).

- We collected  20,000 images on a Titan Krios operating at 300kV (Flacon 4i) yielding about 200k ptcls after 2D classifications and 92,400 ptcls in the 'final' map

When we mentioned 'noise in the map' is that at 9.5A we see some features that we do not think are coming from the protein. Indeed it is possible that we are experiencing  over-sharpening (pls see FSC plot attached).

Can you suggest us some parameters that can be important to increase the map quality (in Cryosparc)?

Thanks again,

Francesca and Sophia













---------------------------------
Francesca Gubellini, PhD
Institut Pasteur
Unité de Microbiologie Structurale
Bat. Metchnikoff
25 rue du Dr Roux
75015 Paris
France
Tel: +33 1 40 61 39 84
----------------------------------


> On 15 Mar 2024, at 00:11, Takanori Nakane <tnakane.protein at osaka-u.ac.jp> wrote:
> 
> Hi,
> 
> > we have a question concerning post-processing of pctls from
> > carbon-coated grids.
> >
> > We see a lot of noise in our map.
> 
> We need more information!
> 
> What do you mean by "a lot of noise"?
> Which software did you use?
> Was the map bad from the beginning, or did it become
> bad after a certain step of processing?
> 
> Does the FSC curve look reasonable?
> Isn't the map over-sharpened?
> If there are huge variations in the local resolution,
> one number might not be adequate.
> 
> In general, there is nothing special about carbon-coated grids.
> 
> Best regards,
> 
> Takanori Nakane
> 
> On 3/15/24 01:17, Francesca GUBELLINI wrote:
>> Dear EM community,
>> we have a question concerning post-processing of pctls from carbon-coated grids.
>> We see a lot of noise in our map.
>> Anyone has specific tips on how to process this kind of data?
>> Thanks a lot!
>> Francesca and Sophia
>>  ---------------------------------
>> Francesca Gubellini, PhD
>> Institut Pasteur
>> Unité de Microbiologie Structurale
>> Bat. Metchnikoff
>> 25 rue du Dr Roux
>> 75015 Paris
>> France
>> Tel: +33 1 40 61 39 84
>> ----------------------------------
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