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<p class="MsoNormal"><span lang="EN-US" style="mso-fareast-language: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 lang="EN-US" style="mso-fareast-language: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 lang="EN-US" style="mso-fareast-language: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 lang="EN-US" style="mso-fareast-language: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 lang="EN-US" style="font-family:"Segoe UI Emoji",sans-serif;mso-fareast-language:EN-US">😊</span><span lang="EN-US" style="mso-fareast-language:EN-US"><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="en-CH" style="mso-fareast-language:EN-US"><o:p> </o:p></span></p>
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<p class="MsoNormal"><b><span lang="EN-US">From:</span></b><span lang="EN-US"> Lucia <luciatorressanchez3@gmail.com>
<br>
<b>Sent:</b> Thursday, 22 April 2021 12:57<br>
<b>To:</b> Marino Jacopo (PSI) <jacopo.marino@psi.ch><br>
<b>Cc:</b> 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> Re: [3dem] Relion issue Autopick - 2D classification<o:p></o:p></span></p>
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<p class="MsoNormal">Hi Jacopo,<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">I try but relion is my nightmare. I feel so useless with it.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<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>
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<p class="MsoNormal"><img width="564" height="306" style="width:5.8697in;height:3.1875in" id="Picture_x0020_1" src="cid:image001.png@01D73779.7A48F090"><o:p></o:p></p>
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<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>
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<p class="MsoNormal"><img width="563" height="342" style="width:5.8645in;height:3.5625in" id="Picture_x0020_2" src="cid:image002.png@01D73779.7A48F090"><o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<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>
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<p class="MsoNormal"><img width="563" height="416" style="width:5.8645in;height:4.3333in" id="Picture_x0020_3" src="cid:image003.png@01D73779.7A48F090"><o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<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>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Thanks!!!!<o:p></o:p></p>
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<p class="MsoNormal">Lucia<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">El jue, 22 abr 2021 a las 12:43, Marino Jacopo (PSI) (<<a href="mailto:jacopo.marino@psi.ch">jacopo.marino@psi.ch</a>>) escribió:<o:p></o:p></p>
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<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>
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<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">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">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>
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<p class="MsoNormal" style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span lang="en-CH"> <o:p></o:p></span></p>
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<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>
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<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>
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