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

Fri Mar 15 08:50:52 PDT 2024

Dynamic masking often does not work very well in cryosparc at low resolution. It may help to tune the dynamic mask settings, or generate your own mask.

HTH,

Axel
________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Guillaume Gaullier <guillaume.gaullier at kemi.uu.se>
Sent: Friday, March 15, 2024 10:45 AM
To: Francesca GUBELLINI <fgubelli at pasteur.fr>
Cc: 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Post-processing of dataset from carbon-coated grids: noise pb

Hello Francesca,

To determine whether you are seeing over-sharpening or something else, compare the unfiltered/unsharpened map and the post-processed map. Their respective filenames end in _map and _map_sharp, among all the output files you can download from the refinement job in cryoSPARC.

At this resolution I would not expect to see noise speckles in the unfiltered/unsharpened map (at a contour level adequate to visualize the protein region).

I hope this helps,

Guillaume

________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Francesca GUBELLINI <fgubelli at pasteur.fr>
Sent: Friday, March 15, 2024 4:03:56 PM
To: Takanori Nakane
Cc: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Post-processing of dataset from carbon-coated grids: noise pb

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

[cid:0648DC20-7E29-4F93-A473-D831D98F9479 at corp.pasteur.fr]

---------------------------------
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<mailto: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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