align particles with a reference image

[andyse...@gmail.com]

Dear All,

I have been using EMAN2 and CryoSPARC for a SPA of a phage protein complex. I need to align the particle stack to a reference image. The particles are extracted out of cryoSPARC workflow and are in mrc format. What will be the command to carry out the process.

Thanks

Andy

Texas A&M University 

[Steve Ludtke]

You want to align a stack of particles to a single reference?  ie - all of the particles are in the same 3-D orientation and only need to be aligned rotationally/translationally in-plane?   Very different if you're also including the orientation determination.

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[Sen, Anindito]

Hi Steve,

All the particles in the stack boxed out from the micrographs need to be aligned to a 2D reference image.  orientation determination will not be needed (something like align2d in EMAN). 

Regards 

Andy 

Regards

Anindito Sen. Ph.D

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From: em...@googlegroups.com <em...@googlegroups.com> on behalf of Steve Ludtke <slud...@gmail.com>
Sent: Sunday, October 19, 2025 9:39 PM
To: em...@googlegroups.com <em...@googlegroups.com>
Subject: Re: [EMAN2] align particles with a reference image

 

You want to align a stack of particles to a single reference? ie - all of the particles are in the same 3-D orientation and only need to be aligned rotationally/translationally in-plane? Very different if you're also including the orientation

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[Steve Ludtke]

Hi Andy,

e2align2d.py

would be the logical place to start. I suspect nobody ever bothered parallelizing it, but unless you have a massive number of particles I wouldn't expect it to take very long regardless. You'll need to specify which aligner you want to use with --align (e2help.py aligners -v2), and which similarity metric you wish to use (e2help.py cmps -v2), though some of the aligners ignore the --aligncmp option. If you want the best results, at the cost of speed, you may also want to use the second stage --ralign (usually refine). I believe you'll get more feedback if you run the program with -v1, but didn't check.

On Oct 20, 2025, at 7:40 AM, 'Sen, Anindito' via EMAN2 <em...@googlegroups.com> wrote:

Hi Steve,

All the particles in the stack boxed out from the micrographs need to be aligned to a 2D reference image.  orientation determination will not be needed (something like align2d in EMAN). 

Regards 

Andy 

Regards

Anindito Sen. Ph.D

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From: em...@googlegroups.com <em...@googlegroups.com> on behalf of Steve Ludtke <slud...@gmail.com>
Sent: Sunday, October 19, 2025 9:39 PM
To: em...@googlegroups.com <em...@googlegroups.com>
Subject: Re: [EMAN2] align particles with a reference image

 

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[Steve Ludtke]

It might be useful to add that the default aligner used for class-averaging is:

rotate_translate_flip:usebispec=1

with the

ccc

similarity metric

and align

refine

with the 

frc:snrweight=1:zeromask=1:minres=80:maxres={}

similarity metric, where the {} are replaced with the maximum resolution to consider in Å