<div dir="auto">Dear Clara, Raj & others<div dir="auto"><br></div><div dir="auto">A discussion on particle picking would be incomplete without a caution against reference bias.</div><div dir="auto"><br></div><div dir="auto">1) be sure to manually inspect your results - can you see your particles? </div><div dir="auto"><br></div><div dir="auto">2) if you skip Takanori's (good) advice and instead use a pdb as Ali had suggested, please try to use a reference that shares low resolution features with your target molecule, but is expected to differ are high res. This way you have a negative control for reference bias & can avoid your own GP160 fiasco, described below</div><div dir="auto"><br></div><div dir="auto">(<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831464/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831464/</a>)</div><div dir="auto"><br></div><div dir="auto">Cheers,</div><div dir="auto"><br></div><div dir="auto">Ben H.</div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Thu, Feb 13, 2020, 11:18 AM <<a href="mailto:3dem-request@ncmir.ucsd.edu">3dem-request@ncmir.ucsd.edu</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">Send 3dem mailing list submissions to<br>
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Today's Topics:<br>
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1. Re: Autopick in relion 3.06 using 3D reference (Ali Punjani)<br>
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Message: 1<br>
Date: Thu, 13 Feb 2020 12:17:27 -0500<br>
From: Ali Punjani <<a href="mailto:alipunjani@cs.toronto.edu" target="_blank" rel="noreferrer">alipunjani@cs.toronto.edu</a>><br>
To: Clara Kielkopf <<a href="mailto:ckielkop@gmail.com" target="_blank" rel="noreferrer">ckielkop@gmail.com</a>><br>
Cc: <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank" rel="noreferrer">3dem@ncmir.ucsd.edu</a><br>
Subject: Re: [3dem] Autopick in relion 3.06 using 3D reference<br>
Message-ID:<br>
<<a href="mailto:CAP_b-V-fSLd_WoXzLYN2vQivXMEPFaR-pSh6tq_pyxvozUTB-w@mail.gmail.com" target="_blank" rel="noreferrer">CAP_b-V-fSLd_WoXzLYN2vQivXMEPFaR-pSh6tq_pyxvozUTB-w@mail.gmail.com</a>><br>
Content-Type: text/plain; charset="utf-8"<br>
<br>
Hi Clara,<br>
<br>
In cryoSPARC, particle picking using references (2D or 3D) does not require<br>
references to be on the same greyscale as the data. You can use a 3D model<br>
generated from a PDB file without issue. The exact steps you might use in<br>
this case are:<br>
<br>
1. Create a volume from PDB (using eg. Chimera)<br>
2. Import the volume into cryoSPARC using the "Import Volume" job<br>
3. Use the ?Create Templates? job to generate 2D templates from various<br>
orientations based on the input volume<br>
4. Use the "Template Picker" job (GPU accelerated) to perform template<br>
picking using the generated references.<br>
<br>
The resulting particle picking scores can be thresholded to cleanly<br>
separate good particle picks from bad, interactively using the "Inspect<br>
Particle Picks" job (gif below):<br>
<br>
[image: ezgif.com-optimize.gif]<br>
<br>
Particle picking is described in the the standard cryoSPARC tutorial (<br>
<a href="https://cryosparc.com/docs/tutorials/t20s/#step-9" rel="noreferrer noreferrer" target="_blank">https://cryosparc.com/docs/tutorials/t20s/#step-9</a>) and more details are<br>
provided in a blog post (<br>
<a href="https://cryosparc.com/docs/tutorials/particle-picking-calibration/" rel="noreferrer noreferrer" target="_blank">https://cryosparc.com/docs/tutorials/particle-picking-calibration/</a>)<br>
<br>
There are also several posts on the cryoSPARC discussion forum (<br>
<a href="https://discuss.cryosparc.com" rel="noreferrer noreferrer" target="_blank">https://discuss.cryosparc.com</a>) regarding strategies and results using<br>
particle picking that may be of interest.<br>
<br>
Hope this helps!<br>
<br>
Thanks,<br>
Ali<br>
<br>
<br>
On Wed, Feb 12, 2020 at 3:14 PM Clara Kielkopf <<a href="mailto:ckielkop@gmail.com" target="_blank" rel="noreferrer">ckielkop@gmail.com</a>> wrote:<br>
<br>
> Takanori,<br>
> Do you have similar reservations about reference-based particle picking in<br>
> Cryosparc?<br>
> Many thanks,<br>
> -Clara<br>
><br>
> Message:<br>
>> Date: Wed, 12 Feb 2020 16:00:45 -0000<br>
>> From: "Takanori Nakane" <<a href="mailto:tnakane@mrc-lmb.cam.ac.uk" target="_blank" rel="noreferrer">tnakane@mrc-lmb.cam.ac.uk</a>><br>
>> To: "Raja Dey" <<a href="mailto:rddey80@gmail.com" target="_blank" rel="noreferrer">rddey80@gmail.com</a>><br>
>> Cc: <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank" rel="noreferrer">3dem@ncmir.ucsd.edu</a><br>
>> Subject: Re: [3dem] Autopick in relion 3.06 using 3D reference<br>
>> Message-ID:<br>
>> <<a href="mailto:5bf02f0ee0bad565b4d4fcb5f87b2f69.squirrel@mail.mrc-lmb.cam.ac.uk" target="_blank" rel="noreferrer">5bf02f0ee0bad565b4d4fcb5f87b2f69.squirrel@mail.mrc-lmb.cam.ac.uk</a><br>
>> ><br>
>> Content-Type: text/plain;charset=iso-2022-jp<br>
>><br>
>> Hi,<br>
>><br>
>> I don't recommend this. I always start by picking<br>
>> about 10K - 100K particles by Laplacian-of-Gaussian picker<br>
>> (this is very quick) and running Class2D to make a<br>
>> template.<br>
>><br>
>> A 3D map from a PDB file does not necessarily match<br>
>> the absolute grey scale of the micrographs. It might work,<br>
>> but not always.<br>
>><br>
>> Best regards,<br>
>><br>
>> Takanori Nakane<br>
>><br>
>> > Dear Members,<br>
>> > I am trying to pick particles using a 3D reference created from a<br>
>> > published<br>
>> > pdb file. I followed these steps to create the reference.<br>
>> > 1. used chimera to create a 15A mrc file from the pdb file.<br>
>> > 2. used relion image handler to change the pixel size (equal to the<br>
>> > micrographs) and box size (approximately double the particle size).<br>
>> > 3. So far, no particle has been picked up after multiple trials<br>
>> > 4. Then I ran the same job as a control on tutorial data of beta<br>
>> > galatosidase and unfortunately no success.<br>
>> ><br>
>> > Don't know what I am missing. If anyone got success and have experience<br>
>> on<br>
>> > running autopicking job in relion3 using 3D reference, sharing your<br>
>> > knowledge would be very helpful.<br>
>> ><br>
>> > Thanks and have a wonderful day.<br>
>> ><br>
>> > Raj<br>
>> > _______________________________________________<br>
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>> ><br>
>><br>
>><br>
>> End of 3dem Digest, Vol 150, Issue 22<br>
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>><br>
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</blockquote></div>