[3dem] Autopick in relion 3.06 using 3D reference

[Ali Punjani]

Hi Clara,

In cryoSPARC, particle picking using references (2D or 3D) does not require
references to be on the same greyscale as the data. You can use a 3D model
generated from a PDB file without issue. The exact steps you might use in
this case are:

   1. Create a volume from PDB (using eg. Chimera)
   2. Import the volume into cryoSPARC using the "Import Volume" job
   3. Use the “Create Templates” job to generate 2D templates from various
   orientations based on the input volume
   4. Use the "Template Picker" job (GPU accelerated) to perform template
   picking using the generated references.

The resulting particle picking scores can be thresholded to cleanly
separate good particle picks from bad, interactively using the "Inspect
Particle Picks" job (gif below):

[image: ezgif.com-optimize.gif]

Particle picking is described in the the standard cryoSPARC tutorial (
https://cryosparc.com/docs/tutorials/t20s/#step-9) and more details are
provided in a blog post (
https://cryosparc.com/docs/tutorials/particle-picking-calibration/)

There are also several posts on the cryoSPARC discussion forum (
https://discuss.cryosparc.com) regarding strategies and results using
particle picking that may be of interest.

Hope this helps!

Thanks,
Ali


On Wed, Feb 12, 2020 at 3:14 PM Clara Kielkopf <ckielkop at gmail.com> wrote:

> Takanori,
> Do you have similar reservations about reference-based particle picking in
> Cryosparc?
> Many thanks,
> -Clara
>
> Message:
>> Date: Wed, 12 Feb 2020 16:00:45 -0000
>> From: "Takanori Nakane" <tnakane at mrc-lmb.cam.ac.uk>
>> To: "Raja Dey" <rddey80 at gmail.com>
>> Cc: 3dem at ncmir.ucsd.edu
>> Subject: Re: [3dem] Autopick in relion 3.06 using 3D reference
>> Message-ID:
>>         <5bf02f0ee0bad565b4d4fcb5f87b2f69.squirrel at mail.mrc-lmb.cam.ac.uk
>> >
>> Content-Type: text/plain;charset=iso-2022-jp
>>
>> Hi,
>>
>> I don't recommend this. I always start by picking
>> about 10K - 100K particles by Laplacian-of-Gaussian picker
>> (this is very quick) and running Class2D to make a
>> template.
>>
>> A 3D map from a PDB file does not necessarily match
>> the absolute grey scale of the micrographs. It might work,
>> but not always.
>>
>> Best regards,
>>
>> Takanori Nakane
>>
>> > Dear Members,
>> > I am trying to pick particles using a 3D reference created from a
>> > published
>> > pdb file. I followed these steps to create the reference.
>> > 1. used chimera to create a 15A mrc file from the pdb file.
>> > 2. used relion image handler to change the pixel size (equal to the
>> > micrographs) and box size (approximately double the particle size).
>> > 3. So far, no particle has been picked up after multiple trials
>> > 4. Then I ran the same job as a control on tutorial data of beta
>> > galatosidase and unfortunately no success.
>> >
>> > Don't know what I am missing. If anyone got success and have experience
>> on
>> > running autopicking job in relion3 using 3D reference, sharing your
>> > knowledge would be very helpful.
>> >
>> > Thanks and have a wonderful day.
>> >
>> > Raj
>> > _______________________________________________
>> > 3dem mailing list
>> > 3dem at ncmir.ucsd.edu
>> > https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>> >
>>
>>
>> End of 3dem Digest, Vol 150, Issue 22
>> *************************************
>>
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