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Hi Lucia,
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<div class="">Sorry to hear about your troubles.</div>
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<div class="">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).</div>
<div class=""><br class="">
</div>
<div class="">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.</div>
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<div class="">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.</div>
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<div class="">I hope this helps, good luck with your project.</div>
<div class="">Cheers,</div>
<div class="">
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<br class="Apple-interchange-newline">
Guillaume</div>
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<div class="">On 22 Apr 2021, at 12:57, Lucia <<a href="mailto:luciatorressanchez3@gmail.com" class="">luciatorressanchez3@gmail.com</a>> wrote:</div>
<br class="Apple-interchange-newline">
<div class="">
<div dir="ltr" class="">Hi Jacopo,
<div class=""><br class="">
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<div class="">I try but relion is my nightmare. I feel so useless with it.</div>
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<div class="">In cryosparc I get nice 2D classes (or that what people say because of course I'm far far from have basic knowledge):</div>
<span id="cid:ii_knsrkfsw0"><Screenshot 2021-04-22 at 12.50.05.png></span><br class="">
<div class="">But then in the 3D, anything makes sense. The "best" AB-initio for the moment looks like...</div>
<div class=""><span id="cid:ii_knsrs8et2"><Screenshot 2021-04-22 at 12.56.00.png></span><br class="">
</div>
<div class=""><br class="">
</div>
<div class=""><br class="">
</div>
<div class="">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:</div>
<div class=""><span id="cid:ii_knsrozm41"><Screenshot 2021-04-22 at 12.53.48.png></span><br class="">
</div>
<div class=""><br class="">
</div>
<div class="">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.</div>
<div class=""><br class="">
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<div class="">Thanks!!!!</div>
<div class="">Lucia</div>
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<br class="">
<div class="gmail_quote">
<div dir="ltr" class="gmail_attr">El jue, 22 abr 2021 a las 12:43, Marino Jacopo (PSI) (<<a href="mailto:jacopo.marino@psi.ch" class="">jacopo.marino@psi.ch</a>>) escribió:<br class="">
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<p class="MsoNormal"><span lang="EN-US" class="">Hi Lucia,<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span lang="EN-US" class="">Don’t be desperate. You are in the initial phase of learning how to use Relion and cryosparc, so frustration might be normal.<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span lang="EN-US" class=""><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span lang="EN-US" class="">Preferential orientation is an interesting topic and it might not impede that you reach high resolution density maps.
<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span lang="EN-US" class="">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.<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span lang="EN-US" class=""><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span lang="EN-US" class="">You can also continue with cryosparc, maybe change the picking parameters to see whether you really don’t have much other 2D classes.<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span lang="EN-US" class=""><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span lang="EN-US" class="">Go to ab-initio with your best 2D-classes. What do you get there ?<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span lang="EN-US" class=""><u class=""></u> <u class=""></u></span></p>
<p class="MsoNormal"><span lang="EN-US" class="">Good luck<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span lang="EN-US" class="">Jacopo<u class=""></u><u class=""></u></span></p>
<p class="MsoNormal"><span lang="en-CH" class=""><u class=""></u> <u class=""></u></span></p>
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<p class="MsoNormal"><b class=""><span lang="EN-US" class="">From:</span></b><span lang="EN-US" class=""> 3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu" target="_blank" class="">3dem-bounces@ncmir.ucsd.edu</a>>
<b class="">On Behalf Of </b>Lucia<br class="">
<b class="">Sent:</b> Thursday, 22 April 2021 12:17<br class="">
<b class="">To:</b> <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank" class="">
3dem@ncmir.ucsd.edu</a><br class="">
<b class="">Subject:</b> [3dem] Relion issue Autopick - 2D classification<u class=""></u><u class=""></u></span></p>
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<p class="MsoNormal"><u class=""></u> <u class=""></u></p>
<div class="">
<p class="MsoNormal">Hi all,<br class="">
<br class="">
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.<u class=""></u><u class=""></u></p>
<div class="">
<p class="MsoNormal"><br class="">
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.<br class="">
<br class="">
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).<br class="">
<br class="">
I was hoping someone could give me some suggestions that help me to overcome that point because I'm desperate.<br class="">
<br class="">
Thanks!<br class="">
Lucia<u class=""></u><u class=""></u></p>
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