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goetschius / isecc

Icosahedral Subparticle Extraction & Correlated Classification
GNU General Public License v3.0
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Unrecognized arguments #3

Open JustasR1 opened 3 years ago

JustasR1 commented 3 years ago

Hi,

I'm trying to get this code to work for a VLP my lab is working on. We are using our own processing system which is running Centos 7. I'm using Miniconda and have a virtual environment setup running python 3.5 with the required programs and their specific versions installed. However, when I try to run the subparticle_extract.py script with the test.star file, I get the error:

ISECC_star_subparticle_extract.py: error: unrecognized arguments: --o --in_parts Import/job012/test.star --j 1

I'm not sure how actively you are monitoring this github or working with the code still, but any help would be appreciated. Thank you!

Best, Justas

goetschius commented 3 years ago

Can you send me the exact command (with arguments) that you used?

For example, on our debian systems:

$ module load isecc/current

$ /opt/python/isecc/bin/python3.5 /opt/isecc/current/ISECC_star_subparticle_extract.py my_input.star --roi threefold --vector 63.794 0 167 --subpart_box 200 --supersym I1 --testmode true

Supply the following arguments:

  • Input star file (must be RELION/3.1 format)
  • Region of interest (fivefold, threefold, twofold, or fullexpand)
  • Vector
  • Subparticle box size
  • Symmetry you refined with (I1)
  • Run with --testmode true to check your parameters!

Where --roi is your region of interest (twofold, threefold, fivefold, or fullexpand), --vector is your subparticle center in x y z, --subpart_box is your desired subparticle box size, --supersym is the global particle symmetry (only I1 is supported currently), and --testmode provides the opportunity to generate an initial model from a small subset of particles in order to more rapidly optimize chosen parameters.

Unless you have a specific reason to use my program, Localized Reconstruction is a great option to consider as well ( https://doi.org/10.1016/j.pbiomolbio.2020.05.004). It's been fully incorporated into Scipion now, so it will likely have strong developer support for the foreseeable future.

I'm in my medical school clinical clerkships at the moment so I can't promise a prompt response, but hopefully this helps.

All the best,

-Dan

On Thu, Apr 8, 2021 at 5:13 PM JustasR1 @.***> wrote:

Hi,

I'm trying to get this code to work for a VLP my lab is working on. We are using our own processing system which is running Centos 7. I'm using Miniconda and have a virtual environment setup running python 3.5 with the required programs and their specific versions installed. However, when I try to run the subparticle_extract.py script with the test.star file, I get the error:

ISECC_star_subparticle_extract.py: error: unrecognized arguments: --o --in_parts Import/job012/test.star --j 1

I'm not sure how actively you are monitoring this github or working with the code still, but any help would be appreciated. Thank you!

Best, Justas

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JustasR1 commented 3 years ago

Thank you for the response, but luckily I don't think anymore troubleshooting is needed! I had been trying to run the script through the External scripts option in Relion 3.1, which was putting in the extra --o argument the program didn't understand. I'm running it through cmd line now, and after a little work with getting the file system correct, it's running.

I'll also take a look at scipion and the localized reconstruction, thanks for the recommendation.

One question I do have, although trial and error would probably fix this for me, is that the program seems to not like the vector I give it that I take from ChimeraX (although you mention that the program should convert A to px)? Our particles symmetry is tricky though, it's a T=4 Chikungunya virus that displays 80 spikes, so that might be why the coordinates for a spike center arn't considered correct. If you have any words of advice they would be appreciated, although no worries and no rush!

Thanks again! Best, Justas

goetschius commented 3 years ago

Determining your vector

Open in Chimera, center your map

(You want your origin index to NOT be 0)

[image: image.png]

Open Volume Tracer

[image: image.png]

Place marker (yellow) centered within your density, within the ASU noted in red.

[image: image.png]

Open the chimera commandline.

With your yellow marker selected, type: getcrd sel (see Figure N.N)

This is your vector.

[image: image.png]

[image: image.png]

Special Vectors

FIVEFOLD: x,y,z should be in the ratio 0 : 0.618 : 1

THREEFOLD: x,y,z should be in the ratio 0.382 : 0 : 1

TWOFOLD: x,y,z should be in the ratio 0 : 0 : 1

The user should idealize their vector to meet the ratios listed above for the fivefold, threefold, or twofold symmetry axis. Internally, ISECC runs a check to confirm a valid vector was chosen, however, the user is ultimately responsible for ensuring that the correct values were used.

Example

An example command with output is shown in figure N.N

Example:

$ /opt/python/isecc/bin/python3.5 /opt/isecc/current/ISECC_star_subparticle_extract.py my_input.star --roi threefold --vector 63.794 0 167 --fudge 1.05 --subpart_box 260 --supersym I1 --testmode true

Initializing.

Note: Sign of local defocus adjustment has been corrected as of 20 Dec 2019.

Threefolds are tricky. Ensure that [ 63.794 0. 167. ] is the vector you want.

Running checkVector

Vector is valid for roi threefold and symmetry I1

Note: Batch mode will be used to speed subparticle generation in relion.

Note: Requested batch size is 3000

Making nearest-vertex assignments for all symops.

Vertex assignments complete!

Applying symmetry rotation 1.000 +0.000i +0.000j +0.000k ( 1 of 20 ) ( 3f01 )

Applying symmetry rotation 0.000 +1.000i +0.000j +0.000k ( 2 of 20 ) ( 3f02 )

Applying symmetry rotation 0.809 -0.500i +0.000j +0.309k ( 3 of 20 ) ( 3f03 )

Applying symmetry rotation -0.309 +0.809i +0.000j -0.500k ( 4 of 20 ) ( 3f04 )

Applying symmetry rotation 0.309 +0.809i +0.000j -0.500k ( 5 of 20 ) ( 3f05 )

Applying symmetry rotation 0.809 +0.500i +0.000j -0.309k ( 6 of 20 ) ( 3f06 )

Applying symmetry rotation -0.500 +0.809i +0.309j +0.000k ( 7 of 20 ) ( 3f07 )

Applying symmetry rotation 0.500 +0.809i +0.309j +0.000k ( 8 of 20 ) ( 3f08 )

Applying symmetry rotation 0.500 +0.809i -0.309j +0.000k ( 9 of 20 ) ( 3f09 )

Applying symmetry rotation 0.809 +0.309i -0.500j +0.000k ( 10 of 20 ) ( 3f10 )

Applying symmetry rotation 0.809 +0.309i +0.500j +0.000k ( 11 of 20 ) ( 3f11 )

Applying symmetry rotation 0.809 -0.309i -0.500j +0.000k ( 12 of 20 ) ( 3f12 )

Applying symmetry rotation 0.809 -0.309i +0.500j +0.000k ( 13 of 20 ) ( 3f13 )

Applying symmetry rotation -0.500 +0.809i -0.309j +0.000k ( 14 of 20 ) ( 3f14 )

Applying symmetry rotation 0.000 +0.809i +0.500j -0.309k ( 15 of 20 ) ( 3f15 )

Applying symmetry rotation 0.500 +0.500i +0.500j -0.500k ( 16 of 20 ) ( 3f16 )

Applying symmetry rotation 0.809 +0.000i +0.309j -0.500k ( 17 of 20 ) ( 3f17 )

Applying symmetry rotation 0.809 -0.500i +0.000j -0.309k ( 18 of 20 ) ( 3f18 )

Applying symmetry rotation 0.809 +0.500i +0.000j +0.309k ( 19 of 20 ) ( 3f19 )

Applying symmetry rotation -0.500 +0.500i -0.500j -0.500k ( 20 of 20 ) ( 3f20 )

Wrote threefoldsubparticle_alignments.star

Subparticle centers and orientations have successfully been defined in star 3.1 format.

WARNING! Please verify that you refined with I1 symmetry

Note: Test Mode selected. Will only generate ~10k subparticles so you can check initial model.

Executing command: head -n10099 threefoldsubparticle_alignments.star > threefoldsubparticle_alignments_abbrev.star

Executing command: relion_stack_create --i threefoldsubparticle_alignments_abbrev.star --o threefold --apply_rounded_offsets_only --split_per_micrograph > /dev/null

NOTE: This will take some time...

         ...isn't it a nice day for a bike ride?

Executing command: relion_image_handler --i threefold.star --o subpart --new_box 260

NOTE: the input (--i) is a STAR file but the output (--o) does not have .mrcs extension. The output is treated as a suffix, not a path.

17/ 17 sec ............................................................~~(,_,">

Written out new STAR file: threefold_subpart.star

Executing command: rm threefold_subparticles/threefold_20200915_1621/Micrographs/batch??????.mrcs

Executing command: head -n10000 threefold_subpart.star > threefold_initialmodel.star

Generating initial model

Executing command: relion_reconstruct --i threefold_initialmodel.star --o threefold_initialmodel_c3.mrc --ctf --maxres 10 --sym c3

Executing command: cp threefold_subpart.star threefold_subpart_PRIOR.star

Executing command: sed -i 's/_rlnAnglePsi/_rlnAnglePsiPrior/g' threefold_subpart_PRIOR.star

Executing command: sed -i 's/_rlnAngleRot/_rlnAngleRotPrior/g' threefold_subpart_PRIOR.star

Executing command: sed -i 's/_rlnAngleTilt/_rlnAngleTiltPrior/g' threefold_subpart_PRIOR.star

Executing command: sed -i 's/_rlnOriginX/_rlnOriginXPrior/g' threefold_subpart_PRIOR.star

Executing command: sed -i 's/_rlnOriginY/_rlnOriginYPrior/g' threefold_subpart_PRIOR.star

NOTE: Moving files to output directory: threefold_subparticles/threefold_20200915_1621/

Success!

Known bugs

Bug001: Fails to create subparticles (2020.Sept.18)

Symptom:

Error along the lines of "Out of space" from relion, but you have plenty of space on disk

the subdirectory fullexpand_subparticles/fullexpand_20200918_0956/ Micrographs was not created

Instead you have a fullexpand_subparticles/fullexpand_20200918_0956/ Micrographs.mrcs file (or similar)

Underlying problem:

The _rlnImageName field in your input star is much shorter than anticipated.

for example, 000025@Particles/0024_new_particles_autopick.mrcs

When ISECC tries to make the _rlnMicrographName field, it can't fit into the array

Instead, it gets cropped to the length of the input star file _rlnImageName

Workaround:

Open your input star file, and pad the first entry's _rlnImageName with extra zeros

for example, 000025@Particles/0024_new_particles_autopick.mrcs

becomes 0000000000000000000000000000000025@Particles /0024_new_particles_autopick.mrcs

Hacky, but effective.

Bug status:

Rare. Not planning to fix.

Preventing this rare error would require manually setting the dtype of the field from the default produced during

my_ndarray = np.asarray( stardata, order='C' )

On Fri, Apr 9, 2021 at 2:22 PM JustasR1 @.***> wrote:

Thank you for the response, but luckily I don't think anymore troubleshooting is needed! I had been trying to run the script through the External scripts option in Relion 3.1, which was putting in the extra --o argument the program didn't understand. I'm running it through cmd line now, and after a little work with getting the file system correct, it's running.

I'll also take a look at scipion and the localized reconstruction, thanks for the recommendation.

One question I do have, although trial and error would probably fix this for me, is that the program seems to not like the vector I give it that I take from ChimeraX (although you mention that the program should convert A to px)? Our particles symmetry is tricky though, it's a T=4 Chikungunya virus that displays 80 spikes, so that might be why the coordinates for a spike center arn't considered correct. If you have any words of advice they would be appreciated, although no worries and no rush!

Thanks again! Best, Justas

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goetschius commented 3 years ago

Ah, and yes. There’s an argument to change pixel size (—angpix maybe?). 1.1 Å is the default. I almost always used 1.1, so I forget that’s even a relevant parameter to set.

All the best.

On Fri, Apr 9, 2021 at 2:22 PM JustasR1 @.***> wrote:

Thank you for the response, but luckily I don't think anymore troubleshooting is needed! I had been trying to run the script through the External scripts option in Relion 3.1, which was putting in the extra --o argument the program didn't understand. I'm running it through cmd line now, and after a little work with getting the file system correct, it's running.

I'll also take a look at scipion and the localized reconstruction, thanks for the recommendation.

One question I do have, although trial and error would probably fix this for me, is that the program seems to not like the vector I give it that I take from ChimeraX (although you mention that the program should convert A to px)? Our particles symmetry is tricky though, it's a T=4 Chikungunya virus that displays 80 spikes, so that might be why the coordinates for a spike center arn't considered correct. If you have any words of advice they would be appreciated, although no worries and no rush!

Thanks again! Best, Justas

— You are receiving this because you commented.

Reply to this email directly, view it on GitHub https://github.com/goetschius/isecc/issues/3#issuecomment-816872038, or unsubscribe https://github.com/notifications/unsubscribe-auth/AIL5S4GK7FV56EH7QHIEHHTTH5AXDANCNFSM42TWKKVQ .