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<p>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.</p>
<p>Kind regards, Carlos Oscar<br>
</p>
<div class="moz-cite-prefix">El 22/04/2021 a las 13:14, Marino
Jacopo (PSI) escribió:<br>
</div>
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<p class="MsoNormal"><span style="mso-fareast-language:EN-US"
lang="EN-US">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 !
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US"
lang="EN-US">It might be some views are hidden within other
classes. How many rounds of 2D-classes and selection you did
already ?<o:p></o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US"
lang="EN-US">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.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US"
lang="EN-US">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 !
</span><span style="font-family:"Segoe UI
Emoji",sans-serif;mso-fareast-language:EN-US"
lang="EN-US">😊</span><span
style="mso-fareast-language:EN-US" lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US"
lang="en-CH"><o:p> </o:p></span></p>
<div style="border:none;border-top:solid #E1E1E1
1.0pt;padding:3.0pt 0cm 0cm 0cm">
<p class="MsoNormal"><b><span lang="EN-US">From:</span></b><span
lang="EN-US"> Lucia <a class="moz-txt-link-rfc2396E" href="mailto:luciatorressanchez3@gmail.com"><luciatorressanchez3@gmail.com></a>
<br>
<b>Sent:</b> Thursday, 22 April 2021 12:57<br>
<b>To:</b> Marino Jacopo (PSI)
<a class="moz-txt-link-rfc2396E" href="mailto:jacopo.marino@psi.ch"><jacopo.marino@psi.ch></a><br>
<b>Cc:</b> <a class="moz-txt-link-abbreviated" href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><br>
<b>Subject:</b> Re: [3dem] Relion issue Autopick - 2D
classification<o:p></o:p></span></p>
</div>
<p class="MsoNormal"><o:p> </o:p></p>
<div>
<p class="MsoNormal">Hi Jacopo,<o:p></o:p></p>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">I try but relion is my nightmare. I
feel so useless with it.<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">In cryosparc I get nice 2D classes (or
that what people say because of course I'm far far from
have basic knowledge):<o:p></o:p></p>
</div>
<p class="MsoNormal"><img
style="width:5.8697in;height:3.1875in"
id="Picture_x0020_1"
src="cid:part1.65A7D99A.5E520711@cnb.csic.es" class=""
width="564" height="306"><o:p></o:p></p>
<div>
<p class="MsoNormal">But then in the 3D, anything makes
sense. The "best" AB-initio for the moment looks like...<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><img
style="width:5.8645in;height:3.5625in"
id="Picture_x0020_2"
src="cid:part2.69E5BBCD.2CB837F4@cnb.csic.es" class=""
width="563" height="342"><o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">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:<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><img
style="width:5.8645in;height:4.3333in"
id="Picture_x0020_3"
src="cid:part3.0105F47C.E9A87F3C@cnb.csic.es" class=""
width="563" height="416"><o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">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.<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">Thanks!!!!<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">Lucia<o:p></o:p></p>
</div>
</div>
<p class="MsoNormal"><o:p> </o:p></p>
<div>
<div>
<p class="MsoNormal">El jue, 22 abr 2021 a las 12:43, Marino
Jacopo (PSI) (<<a href="mailto:jacopo.marino@psi.ch"
moz-do-not-send="true">jacopo.marino@psi.ch</a>>)
escribió:<o:p></o:p></p>
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<div>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="EN-US">Hi Lucia,</span><span lang="en-CH"><o:p></o:p></span></p>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="EN-US">Don’t be desperate. You are in the
initial phase of learning how to use Relion and
cryosparc, so frustration might be normal.</span><span
lang="en-CH"><o:p></o:p></span></p>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="EN-US"> </span><span lang="en-CH"><o:p></o:p></span></p>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="EN-US">Preferential orientation is an
interesting topic and it might not impede that you
reach high resolution density maps.
</span><span lang="en-CH"><o:p></o:p></span></p>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="EN-US">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.</span><span lang="en-CH"><o:p></o:p></span></p>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="EN-US"> </span><span lang="en-CH"><o:p></o:p></span></p>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="EN-US">You can also continue with cryosparc,
maybe change the picking parameters to see whether
you really don’t have much other 2D classes.</span><span
lang="en-CH"><o:p></o:p></span></p>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="EN-US"> </span><span lang="en-CH"><o:p></o:p></span></p>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="EN-US">Go to ab-initio with your best
2D-classes. What do you get there ?</span><span
lang="en-CH"><o:p></o:p></span></p>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="EN-US"> </span><span lang="en-CH"><o:p></o:p></span></p>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="EN-US">Good luck</span><span lang="en-CH"><o:p></o:p></span></p>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="EN-US">Jacopo</span><span lang="en-CH"><o:p></o:p></span></p>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="en-CH"> <o:p></o:p></span></p>
<div style="border:none;border-top:solid #E1E1E1
1.0pt;padding:3.0pt 0cm 0cm 0cm">
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><b><span
lang="EN-US">From:</span></b><span lang="EN-US">
3dem <<a
href="mailto:3dem-bounces@ncmir.ucsd.edu"
target="_blank" moz-do-not-send="true">3dem-bounces@ncmir.ucsd.edu</a>>
<b>On Behalf Of </b>Lucia<br>
<b>Sent:</b> Thursday, 22 April 2021 12:17<br>
<b>To:</b> <a href="mailto:3dem@ncmir.ucsd.edu"
target="_blank" moz-do-not-send="true">3dem@ncmir.ucsd.edu</a><br>
<b>Subject:</b> [3dem] Relion issue Autopick - 2D
classification</span><span lang="en-CH"><o:p></o:p></span></p>
</div>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="en-CH"> <o:p></o:p></span></p>
<div>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="en-CH">Hi all,<br>
<br>
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.<o:p></o:p></span></p>
<div>
<p class="MsoNormal"
style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span
lang="en-CH"><br>
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>
<br>
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>
<br>
I was hoping someone could give me some
suggestions that help me to overcome that point
because I'm desperate.<br>
<br>
Thanks!<br>
Lucia<o:p></o:p></span></p>
</div>
</div>
</div>
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