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| == Single Particle Tomography in EMAN2 == === Wed PM Practical === |
= Single Particle Tomography in EMAN2 = == Wednesday - P.M. == |
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| SPT (single particle tomography) capabilities are relatively new in EMAN2. They were inspired by Michael Schmid's studies on sub-volume averaging (mostly on viruses), and a stubborn student insisting on doing extensive sub-volume averaging on chaperons. | This session will cover the new tools for single particle tomography in EMAN2. Unfortunately, this technique is very memory-intensive and compute-intensive. The 3 gb of ram available on the workshop computers is barely sufficient to do some small examples, and full 3-D alignments of large sets of particles can take many hours of computation. |
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| This session will cover the beta version of a small fraction of the SPT capabilities EMAN2 will eventually make available. | So, for purposes of the tutorial, we will learn how to use the particle picker, and do a couple of small examples which can be completed in the available time. |
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| The session will cover: 1) SPT processing through EMAN2's workflow: e2workflow.py 2) Sub-volume extraction from tomograms using e2tomoboxer.py 3) "Preparation" of extracted particles for alignment (for a myriad of reasons, it is NOT recommendable to align and average sub-volumes directly after extraction). 4) Reference based alignment and averaging |
== DATA == [[attachment:e2spt_data.zip| e2spt_data.zip|&do=get]] |
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| All necessary software is provided as part of EMAN2. If you don't have EMAN2 installed, you can download the most updated version (for your specific platform, Windows, Linux or Mac), from here: http://ncmi.bcm.edu/ncmi/software/counter_222/software_86 |
== TUTORIAL DOCUMENT == Not available here for now. Get it through this site: |
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| We have prepared sample data for this tutorial. The tomogram in the link below comes from a tilt series of epsilon15 virus particles in vitro, recorded using Zernike phase-plate technology: |
http://blake.bcm.edu/emanwiki/Ws2011/Agenda |
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| [[attachment:e15phaseplate.rec]] | == Monstrous command for alignment with e2classaverage3d.py == |
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| The following tomogram also comes from a tilt series of epsilon15 viruses in vitro but was recorded under conventional cryoEM imaging conditions: | e2classaverage3d.py --input=e15pp_set1_stack.hdf --output=e15pp_set1_aligned.hdf --ref=e15ref_prep_icos_bin2.hdf --npeakstorefine=1 -v 3 --mask=mask.sharp:outer_radius=48 --preprocess=filter.lowpass.gauss:cutoff_freq=.02 --align=rotate_translate_3d:search=10:delta=8:dphi=8:verbose=1:sym=icos --parallel=thread:2 --ralign=refine_3d_grid:delta=3:range=9 --averager=mean.tomo --aligncmp=ccc.tomo --raligncmp=ccc.tomo --shrink=3 --shrinkrefine=3 --savesteps --saveali --iter=8 --normproc=normalize --sym=c1 --keep=0.8 |
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| [[attachment:e15normal.rec]] | == Monstrous command for alignment with e2tomoallvsall.py == e2tomoallvsall.py -v 1 --path=AVSAs087 --input=CENTEREDvsD8aliVSapo_s087.hdf --shrink=3 --shrinkrefine=2 --iter=87 --mask=mask.sharp:outer_radius=36 --npeakstorefine=16 --preprocess=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --align=rotate_translate_3d:search=4:dphi=12:delta=12 --parallel=thread:24 --ralign=refine_3d_grid:delta=3:range=12:search=2 --averager=mean.tomo --aligncmp=ccc.tomo --raligncmp=ccc.tomo --saveali --savesteps -v 2 --postprocess=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --autocenter --exclusive_class_min=8 --normproc=normalize |
Single Particle Tomography in EMAN2
Wednesday - P.M.
This session will cover the new tools for single particle tomography in EMAN2. Unfortunately, this technique is very memory-intensive and compute-intensive. The 3 gb of ram available on the workshop computers is barely sufficient to do some small examples, and full 3-D alignments of large sets of particles can take many hours of computation.
So, for purposes of the tutorial, we will learn how to use the particle picker, and do a couple of small examples which can be completed in the available time.
DATA
TUTORIAL DOCUMENT
Not available here for now. Get it through this site:
http://blake.bcm.edu/emanwiki/Ws2011/Agenda
Monstrous command for alignment with e2classaverage3d.py
e2classaverage3d.py --input=e15pp_set1_stack.hdf --output=e15pp_set1_aligned.hdf --ref=e15ref_prep_icos_bin2.hdf --npeakstorefine=1 -v 3 --mask=mask.sharp:outer_radius=48 --preprocess=filter.lowpass.gauss:cutoff_freq=.02 --align=rotate_translate_3d:search=10:delta=8:dphi=8:verbose=1:sym=icos --parallel=thread:2 --ralign=refine_3d_grid:delta=3:range=9 --averager=mean.tomo --aligncmp=ccc.tomo --raligncmp=ccc.tomo --shrink=3 --shrinkrefine=3 --savesteps --saveali --iter=8 --normproc=normalize --sym=c1 --keep=0.8
Monstrous command for alignment with e2tomoallvsall.py
e2tomoallvsall.py -v 1 --path=AVSAs087 --input=CENTEREDvsD8aliVSapo_s087.hdf --shrink=3 --shrinkrefine=2 --iter=87 --mask=mask.sharp:outer_radius=36 --npeakstorefine=16 --preprocess=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --align=rotate_translate_3d:search=4:dphi=12:delta=12 --parallel=thread:24 --ralign=refine_3d_grid:delta=3:range=12:search=2 --averager=mean.tomo --aligncmp=ccc.tomo --raligncmp=ccc.tomo --saveali --savesteps -v 2 --postprocess=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --autocenter --exclusive_class_min=8 --normproc=normalize