[3dem] Relion issue Autopick - 2D classification

Thu Apr 22 04:29:18 PDT 2021

Hi Lucia,

Sorry to hear about your troubles.

I agree with Jacopo that the suggestion to use a different program to overcome orientation bias of your particles is not very helpful without more specific suggestions. I think your initial 3D model in cryoSPARC looks decent. Have you looked at it in a 3D viewer like ChimeraX? Does it make sense? A good initial model should have connected density (multiple disconnected blobs is not good), and if the overall shape corresponds to what you see in the 2D classes (which seems to be the case here, based on what you showed), you should try to use this initial model to run 3D classification (it’s called "heterogeneous 3D refinement" in cryoSPARC vocabulary). The number of 3D classes to request depends on how much different species you think are present in the set of particles. I recommend starting with a small number of classes like 4, see if the results make sense, and if not try with a larger number of classes (maybe up to 10).

The above suggestions should be easily doable from where you are now in cryoSPARC. That said, if you really are missing orientations, you do need to go back to picking indeed. The most effective way to find particles with rare orientations in the micrographs is to use a neural net particle picker like crYOLO or Topaz. cryoSPARC has an interface to Topaz (I think you still need to install Topaz, and when you have it indicate in cryoSPARC where to find the program), so it’s probably the easiest way to go in your case.

You didn’t say how you performed particle picking in cryoSPARC. I am assuming it was with template matching? (manually pick enough particles to get good 2D classes, then use these 2D classes as references for automated picking). In many published cases and also from my own experience processing a few datasets, 3D reconstructions can improve a lot when picking with Topaz or crYOLO, compared to template matching or LoG. Neural net pickers seem a lot better at not only finding good particles, but also avoiding bad ones, which helps a lot overall (even a small proportion of bad particles sometimes easily messes with 2D and 3D classification, so the most robust way to deal with these is to avoid picking them in the first place). Starting over from picking is a lot of work, so you might as well use the most robust method while you’re at it.

I hope this helps, good luck with your project.
Cheers,

Guillaume

On 22 Apr 2021, at 12:57, Lucia <luciatorressanchez3 at gmail.com<mailto:luciatorressanchez3 at gmail.com>> wrote:

Hi Jacopo,

I try but relion is my nightmare. I feel so useless with it.

In cryosparc I get nice 2D classes (or that what people say because of course I'm far far from have basic knowledge):
<Screenshot 2021-04-22 at 12.50.05.png>
But then in the 3D, anything makes sense. The "best" AB-initio for the moment looks like...
<Screenshot 2021-04-22 at 12.56.00.png>

So I moved to relion and I set different combinations of parameters, but when I try to do the 2D classes (independently of my parameters in the autopick) I get that:
<Screenshot 2021-04-22 at 12.53.48.png>

It's obvious even for me that I'm doing something AWFULLY wrong, but I swear I tried and I don't understand what I'm doing wrong neither how to overcome it.

Thanks!!!!
Lucia

El jue, 22 abr 2021 a las 12:43, Marino Jacopo (PSI) (<jacopo.marino at psi.ch<mailto:jacopo.marino at psi.ch>>) escribió:
Hi Lucia,
Don’t be desperate. You are in the initial phase of learning how to use Relion and cryosparc, so frustration might be normal.

Preferential orientation is an interesting topic and it might not impede that you reach high resolution density maps.
The suggestion of using Relion to circumvent orientation bias is funny. In Relion you might need to play around with the parameters to pick particles, better you do it with a small set of micrographs.

You can also continue with cryosparc, maybe change the picking parameters to see whether you really don’t have much other 2D classes.

Go to ab-initio with your best 2D-classes. What do you get there ?

Good luck
Jacopo

From: 3dem <3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>> On Behalf Of Lucia
Sent: Thursday, 22 April 2021 12:17
To: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
Subject: [3dem] Relion issue Autopick - 2D classification

Hi all,

Sorry for bother and thanks for your patience/help. I’m trying to use Relion to Autopick (Laplacian) a set of elongated particles that are around 150A long. I managed to get a nice set of 2D classes in Cryosparc but, since I have preferential orientation, people recommend me to use Relion to redo my dataset analysis.

But... I'm really stuck, I have no experience with Relion and I don't manage to get any good autopicking that lead me to a 2D classification (even a preliminar one to re do the picking after), I just get black images or nonsense in the 2D classification, even when I check the images after the autopicking and they don't look bad.

Honestly idk what to put here about the parameters that I'm using because I tried a bunch but blindly (since I have no idea neither experience).

I was hoping someone could give me some suggestions that help me to overcome that point because I'm desperate.

Thanks!
Lucia
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