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<p class="MsoNormal"><b>From:</b> Collaborative Computational Project in Electron cryo-Microscopy <CCPEM@JISCMAIL.AC.UK>
<b>On Behalf Of </b>CCPEM automatic digest system<br>
<b>Sent:</b> Friday, July 21, 2023 5:13 AM<br>
<b>To:</b> CCPEM@JISCMAIL.AC.UK<br>
<b>Subject:</b> CCPEM Digest - 21 Jul 2023 - Special issue (#2023-193)<o:p></o:p></p>
</div>
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<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">There is 1 message totaling 27021 lines in this issue.<br>
<br>
Topics in this special issue:<br>
<br>
1. Help w 3D Classification<br>
<br>
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----------------------------------------------------------------------<br>
<br>
Date: Fri, 21 Jul 2023 11:12:31 +0200<br>
From: Bastiaan Bergman <<a href="mailto:bastiaan@MODELMEDICINES.COM">bastiaan@MODELMEDICINES.COM</a>><br>
Subject: Re: Help w 3D Classification<br>
<br>
Takanori, CCPEM,<br>
<br>
Thanks for all your help with this. My dataset has been processed using<br>
Cryospark, creating a 2.3A structure. I'm trying to repeat that with<br>
Relion, but struggle to get anything better than 12.5A. Below an image of<br>
the 2D classes from Cryospark, and the 2D classes I get with Relion. Would<br>
you say one is much better than the other?<br>
<br>
Thanks again for all your help, hints and thoughts!<br>
Bastiaan.<br>
<br>
Cryospark classes:<br>
[image: Screenshot 2023-07-21 at 11.08.28.png]<br>
<br>
top 20 of 100 Relion classes:<br>
[image: Screenshot 2023-07-21 at 11.10.53.png]<br>
<br>
On Fri, Jul 21, 2023 at 12:11 AM Takanori Nakane <<br>
<a href="mailto:tnakane.protein@osaka-u.ac.jp">tnakane.protein@osaka-u.ac.jp</a>> wrote:<br>
<br>
> Hi,<br>
><br>
> Try Class2D with "ignore CTF until first peak: yes".<br>
> Particle picking looks OK.<br>
><br>
> But it is very difficult to tell if your sample<br>
> is good enough (not too heterogeneous, good ice thickness)<br>
> without actually processing it myself. If you are new<br>
> to SPA, you should ask some experts around you to process the<br>
> dataset together. You can also download some good datasets<br>
> from EMPIAR to practice more before processing your<br>
> own harder datasets.<br>
><br>
> Best regards,<br>
><br>
> Takanori Nakane<br>
><br>
> On 7/20/23 23:54, Bastiaan Bergman wrote:<br>
> > Perfect, glad I got this clear now. What can I do in 2D<br>
> > classification to fix this? I'm playing with Regularisation 4 & 8 and<br>
> > limit resolution E-step 7 & 12 now,.. Or should I go further back?<br>
> > Particle picking, CTF, motion correction and EER frame grouping,...<br>
> > Below one micrograph with picked particles, anything that stands out on<br>
> > this?<br>
> ><br>
> > image.png<br>
> > image.png<br>
> ><br>
> ><br>
> ><br>
> ><br>
> ><br>
> > On Thu, Jul 20, 2023 at 4:10 PM Takanori Nakane<br>
> > <<a href="mailto:tnakane.protein@osaka-u.ac.jp%20%3cmailto:tnakane.protein@osaka-u.ac.jp">tnakane.protein@osaka-u.ac.jp <mailto:tnakane.protein@osaka-u.ac.jp</a>>><br>
> > wrote:<br>
> ><br>
> > Hi,<br>
> ><br>
> > Indeed all classes look like noise, unfortunately.<br>
> > Class12 might contain some real particles, but isn't very promising<br>
> > anyway.<br>
> ><br>
> > Best regards,<br>
> ><br>
> > Takanori Nakane<br>
> ><br>
> > On 7/20/23 23:00, Bastiaan Bergman wrote:<br>
> > > Yes, someone else noted that my 2D classes might be noise fitted<br>
> to<br>
> > > itself, see 2d class averages and the initial model below. Maybe<br>
> > that's<br>
> > > where the real problem is. I don't have enough experience to<br>
> really<br>
> > > judge those images, so your view would be very valuable.<br>
> > ><br>
> > > image.png<br>
> > ><br>
> > > job238_initial_model.png<br>
> > ><br>
> > ><br>
> > ><br>
> > > On Thu, Jul 20, 2023 at 2:29 PM Takanori Nakane<br>
> > > <<a href="mailto:tnakane.protein@osaka-u.ac.jp%0b">tnakane.protein@osaka-u.ac.jp<br>
</a>> > <<a href="mailto:tnakane.protein@osaka-u.ac.jp">mailto:tnakane.protein@osaka-u.ac.jp</a>><br>
> > <<a href="mailto:tnakane.protein@osaka-u.ac.jp%0b">mailto:tnakane.protein@osaka-u.ac.jp<br>
</a>> > <<a href="mailto:tnakane.protein@osaka-u.ac.jp">mailto:tnakane.protein@osaka-u.ac.jp</a>>>><br>
> > > wrote:<br>
> > ><br>
> > > Hi,<br>
> > ><br>
> > > Does your initial model show reasonable, protein-like<br>
> features?<br>
> > ><br>
> > > What happens if you run Class3D or Refine3D without a 3D mask?<br>
> > ><br>
> > > Best regards,<br>
> > ><br>
> > > Takanori Nakane<br>
> > ><br>
> > > On 7/20/23 21:24, Bastiaan Bergman wrote:<br>
> > > > Thanks Takanori and Guillaume,<br>
> > > ><br>
> > > > I did let it run, after the first iteration they're all<br>
> > zero and<br>
> > > never<br>
> > > > recover from it.<br>
> > > > See output from relion_image_handler below. This is before<br>
> > > Refine3D. My<br>
> > > > particles are 80-90Angstrom in diameter, I took a mask<br>
> > diameter<br>
> > > of 152<br>
> > > > px and a particle box of 202 px (152 is 75% of 202). I had<br>
> > some<br>
> > > issues<br>
> > > > with neighbouring particles, hence the specific numbers<br>
> here.<br>
> > > ><br>
> > > > relion_image_handler --i Select/job219/particles.star<br>
> --stats<br>
> > > > ---<br>
> > > > <a href="mailto:000007@Extract/job217/Movies/../FoilHole_.._36_EER.mrcs">
000007@Extract/job217/Movies/../FoilHole_.._36_EER.mrcs</a> :<br>
> > > (x,y,z,n)= 202<br>
> > > > x 202 x 1 x 1 ; avg= -0.00126833 stddev= 1.00454 minval=<br>
> > -6.04688<br>
> > > > maxval= 2.83398; angpix = 0.727<br>
> > > ><br>
> > > > relion_image_handler --i<br>
> InitialModel/job238/initial_model.mrc<br>
> > > --stats<br>
> > > > InitialModel/job238/initial_model.mrc : (x,y,z,n)= 202 x<br>
> 202 x<br>
> > > 202 x 1 ;<br>
> > > > avg= 0.000125179 stddev= 0.000766316 minval= -0.00732448<br>
> > maxval=<br>
> > > > 0.00910449; angpix = 0.727<br>
> > > ><br>
> > > > Hope to get some ideas!<br>
> > > > Thanks, Bastiaan.<br>
> > > ><br>
> > > ><br>
> > > ><br>
> > > > On Thu, Jul 20, 2023 at 1:07 PM Takanori Nakane<br>
> > > > <<a href="mailto:tnakane.protein@osaka-u.ac.jp%0b">tnakane.protein@osaka-u.ac.jp<br>
</a>> > <<a href="mailto:tnakane.protein@osaka-u.ac.jp">mailto:tnakane.protein@osaka-u.ac.jp</a>><br>
> > > <<a href="mailto:tnakane.protein@osaka-u.ac.jp%0b">mailto:tnakane.protein@osaka-u.ac.jp<br>
</a>> > <<a href="mailto:tnakane.protein@osaka-u.ac.jp">mailto:tnakane.protein@osaka-u.ac.jp</a>>><br>
> > > <<a href="mailto:tnakane.protein@osaka-u.ac.jp%0b">mailto:tnakane.protein@osaka-u.ac.jp<br>
</a>> > <<a href="mailto:tnakane.protein@osaka-u.ac.jp">mailto:tnakane.protein@osaka-u.ac.jp</a>><br>
> > > <<a href="mailto:tnakane.protein@osaka-u.ac.jp%0b">mailto:tnakane.protein@osaka-u.ac.jp<br>
</a>> > <<a href="mailto:tnakane.protein@osaka-u.ac.jp">mailto:tnakane.protein@osaka-u.ac.jp</a>>>>><br>
> > > > wrote:<br>
> > > ><br>
> > > > Hi,<br>
> > > ><br>
> > > > No, if they are really "all zeros" (please check with<br>
> > > > relion_image_handler --stats), they won't serve as<br>
> > references.<br>
> > > ><br>
> > > > Something is wrong. For example, is the mask adequate?<br>
> > > > Is this after Refine3D or before Refine3D?<br>
> > > ><br>
> > > > Best regards,<br>
> > > ><br>
> > > > Takanori Nakane<br>
> > > ><br>
> > > > On 7/20/23 20:03, Guillaume Gaullier wrote:<br>
> > > > > Hello,<br>
> > > > ><br>
> > > > > Does the job continue running normally after the<br>
> first<br>
> > > iteration?<br>
> > > > If so,<br>
> > > > > I wouldn’t worry about this. It’s highly unlikely<br>
> > (impossible,<br>
> > > > even?)<br>
> > > > > that a 3D classification would converge in the<br>
> > first couple of<br>
> > > > > iterations, so give it a few more iterations until<br>
> > you look at<br>
> > > > the maps,<br>
> > > > > then if they haven’t started taking shapes you can<br>
> > > recognize as<br>
> > > > > consistent with 2D class averages, it might be time<br>
> > to worry.<br>
> > > > ><br>
> > > > > I hope this helps,<br>
> > > > ><br>
> > > > > Guillaume<br>
> > > > ><br>
> > > > ><br>
> > > > >> On 20 Jul 2023, at 10:01, Bastiaan Bergman<br>
> > > > >> <<a href="mailto:bastiaan@MODELMEDICINES.COM%0b">bastiaan@MODELMEDICINES.COM<br>
</a>> > <<a href="mailto:bastiaan@MODELMEDICINES.COM">mailto:bastiaan@MODELMEDICINES.COM</a>><br>
> > > <<a href="mailto:bastiaan@MODELMEDICINES.COM%0b">mailto:bastiaan@MODELMEDICINES.COM<br>
</a>> > <<a href="mailto:bastiaan@MODELMEDICINES.COM">mailto:bastiaan@MODELMEDICINES.COM</a>>><br>
> > > > <<a href="mailto:bastiaan@MODELMEDICINES.COM%0b">mailto:bastiaan@MODELMEDICINES.COM<br>
</a>> > <<a href="mailto:bastiaan@MODELMEDICINES.COM">mailto:bastiaan@MODELMEDICINES.COM</a>><br>
> > > <<a href="mailto:bastiaan@MODELMEDICINES.COM%0b">mailto:bastiaan@MODELMEDICINES.COM<br>
</a>> > <<a href="mailto:bastiaan@MODELMEDICINES.COM">mailto:bastiaan@MODELMEDICINES.COM</a>>>><br>
> > > > <<a href="mailto:bastiaan@MODELMEDICINES.COM%0b">mailto:bastiaan@MODELMEDICINES.COM<br>
</a>> > <<a href="mailto:bastiaan@MODELMEDICINES.COM">mailto:bastiaan@MODELMEDICINES.COM</a>><br>
> > > <<a href="mailto:bastiaan@MODELMEDICINES.COM%0b">mailto:bastiaan@MODELMEDICINES.COM<br>
</a>> > <<a href="mailto:bastiaan@MODELMEDICINES.COM">mailto:bastiaan@MODELMEDICINES.COM</a>>><br>
> > > > <<a href="mailto:bastiaan@MODELMEDICINES.COM%0b">mailto:bastiaan@MODELMEDICINES.COM<br>
</a>> > <<a href="mailto:bastiaan@MODELMEDICINES.COM">mailto:bastiaan@MODELMEDICINES.COM</a>><br>
> > > <<a href="mailto:bastiaan@MODELMEDICINES.COM%0b">mailto:bastiaan@MODELMEDICINES.COM<br>
</a>> > <<a href="mailto:bastiaan@MODELMEDICINES.COM">mailto:bastiaan@MODELMEDICINES.COM</a>>>>>> wrote:<br>
> > > > >><br>
> > > > >> Hi CCPEM-ers,<br>
> > > > >><br>
> > > > >> A noob question: I try to 3d Classify 35000<br>
> particles<br>
> > > (202x202<br>
> > > > pixels<br>
> > > > >> full resolution) in 6 classes. After the first<br>
> > > > iteration classXXX.mrc<br>
> > > > >> files are all zeros. What parameter can I look at<br>
> > to fix<br>
> > > this issue?<br>
> > > > >><br>
> > > > >> Thanks,<br>
> > > > >> Bastiaan.<br>
> > > > >><br>
> > > > >><br>
> > > ><br>
> > ><br>
> ><br>
> ------------------------------------------------------------------------<br>
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> ><br>
><br>
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End of CCPEM Digest - 21 Jul 2023 - Special issue (#2023-193)<br>
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