[3dem] Relion issue Autopick - 2D classification

Thu Apr 22 05:25:41 PDT 2021

Hello Lucia,

From your 2D classification and 3D refinement results in cryosparc, I would say your project is very promissing. You have so many particles and relatively enough views for the 3D reconstruction. I totally agree with Guillaume's suggestions above. What you can do is to build 4 or more  initial models with in cryosparc and use them as references for heterogenous refinement. I guess you will get at least one good class in which the reconstruction is less anisotropic.

Good luck!

Best,

Jun

________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Lucia <luciatorressanchez3 at gmail.com>
Sent: Thursday, April 22, 2021 1:57:29 PM
To: Carlos Oscar Sorzano; pgerlach at biochem.mpg.de; Marino Jacopo (PSI); David.Bhella at glasgow.ac.uk; guillaume.gaullier at icm.uu.se
Cc: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Relion issue Autopick - 2D classification

Hi all!!

Hi Piotr :),

Thanks for your Relion screenshots (very useful because idk most of the commands) and suggestions, I'll try to input them on my 2D classification and see what happens! (Fingers crossed).

Maybe I'll say shit... but as far as I know I don't have Topaz implemented so I'll try to figure it out. But I think that suggestions it's cool not only for me but also for other mates!!!

I'm really grateful for your help!!!

Hi again Jacopo :D,

I think I have some orientation plus heterogenity plus idk what, because I have the crystal structures of most of the domains of my complex but we can't fit anything inside :/. We were hoping better results with that huge batch from the Titan but we're in the same point as we were with the Talos batches.

We performed 5 2D round on Cryosparc.

I let here some screenshots about Relion autopick:
[Screenshot 2021-04-22 at 13.19.01.png]
[Screenshot 2021-04-22 at 13.19.12.png]

And 2D classification:
[Screenshot 2021-04-22 at 13.41.25.png]
[Screenshot 2021-04-22 at 13.41.17.png]
[Screenshot 2021-04-22 at 13.41.33.png]

Thanks a million!!!

Hi David :),

Thanks for the CTF suggestion, I'll definitely try that!
For the TOpaz I don't think I have it. Also I wanted to export from Cryosparc to Relion either my 2D either my preliminar 3D, but when I tried I just got a failed job or a 0 picking in Relion, I guess I did something wrong, but I don't know what.

Thanks a bunch!

Hi Guillaume :3,

People that mention that just said, Relion works better with preferential orientation issues, but since I have no clue about the topic I'm no-one to discuss, and well.. try both in parallel is not a bad idea and it will force me to learn more (but it's being hard!!!).

I did a 3D refinement in cryosparc
[Screenshot 2021-04-22 at 13.49.02.png]
[Screenshot 2021-04-22 at 13.49.10.png]
[Screenshot 2021-04-22 at 13.49.18.png]
But when I look at the chimera model it doesn look ok, I have... slices (it's the best description I can make). That's why they said I have preferential orientation I guess.

For the 3D classes I tried 3 and 4, and it looks ...it can work but then I move to the refinement and no.

I need to figure out how to install topaz and also get green light to do it. But I see everyone is really advision Topaz, so I'll definitely try to get it.

Oh no, In cryosparc I did a blob picking and then with the first 2D classes set I tried to improve my picking using it as a template.

OMG so many good suggestions, I'm really grateful you spent time to help me. THANKS!!!!

Hola Oscar :D,

I never heard about that methods before, I'll try to get more information about them. Thanks!!!

Best,
Lucia

El jue, 22 abr 2021 a las 13:29, Carlos Oscar Sorzano (<coss at cnb.csic.es<mailto:coss at cnb.csic.es>>) escribió:

I agree that it seems you have enough views and maybe the problem is in the generation of the initial volume. You may try with methods that work with class representatives, rather than raw images, in that way you get away from the preferred views. RANSAC and reconstruct significant from Xmipp are ones of these methods. You can access them through Scipion.

Kind regards, Carlos Oscar

El 22/04/2021 a las 13:14, Marino Jacopo (PSI) escribió:
To me it does not look like you have  a severe orientation bias (if you have it at all), and you have plenty of particles !
It might be some views are hidden within other classes. How many rounds of 2D-classes and selection you did already ?
For ab-initio, you might play around with parameters (2 classes, zero similarity, higher number of final iterations, etc.) look on the cryosparc forum for similar discussions. Sometimes you need to play with the ab-initio till you get a volume that makes sense, then take the particles that come with it and go to refinement.
If you want help with the parameters for Relion, you need to post a screenshot of your GUI with the parameters. Relion works well too ! 😊

From: Lucia <luciatorressanchez3 at gmail.com><mailto:luciatorressanchez3 at gmail.com>
Sent: Thursday, 22 April 2021 12:57
To: Marino Jacopo (PSI) <jacopo.marino at psi.ch><mailto:jacopo.marino at psi.ch>
Cc: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Relion issue Autopick - 2D classification

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):
[cid:178f95b9a9a4cff311]
But then in the 3D, anything makes sense. The "best" AB-initio for the moment looks like...
[cid:178f95b9a9a5b16b22]

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:
[cid:178f95b9a9a692e333]

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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