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

[Jan Binovsky]

Dear Raj,

if you insist on using Relion, you can also exploit *relion_project* tool
to generate desired number of backprojections (which will be on absolute
grayscale) of your reference map. Then you can select a few of them for
2D-template-based picking.

Best regards,

Jan


On Thu, Feb 13, 2020, 6:18 PM Ali Punjani <alipunjani at cs.toronto.edu> wrote:

> 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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