|
Size: 5418
Comment:
|
Size: 3620
Comment:
|
| Deletions are marked like this. | Additions are marked like this. |
| Line 2: | Line 2: |
| == Wednesday March 16, 2011 - P.M. == | |
| Line 4: | Line 3: |
| This session will be about extracting sub-volumes from a tomogram, preparing them for alignment, aligning them, and averaging them. The necessary software is provided through EMAN2. All the data you need is inside a .zip file below which contains: a) Two tomograms of epsilon15 virus (e15) b) 3 e15 "references" (an asymmetric one, and one with icosahedral symmetry, both prepared for alignment; and a raw one, straight from the EMDB) c) 1 text file (a small "coordinates file") |
Unfortunately, this technique SPT is very computationally intensive (both in terms of memory and processing speed). 3GB of RAM is the bare minimum recommended to get through the tutorial. For more realistic SPT on full 3D alignments on large sets of large particles particles, 8GB of memory and the use of multiple processing units is advised. |
| Line 11: | Line 7: |
| Download the written tutorial provided through the PDF file below. | |
| Line 13: | Line 8: |
| [The links SHOULD work directly (it is safe to do right-click followed by "save as"); if not, once you click on the link it will take you to another page where you'll see a "DOWNLOAD" link; click on that to get the corresponding file]. | == E2SPT USERS' GUIDE == |
| Line 15: | Line 10: |
| == TUTORIAL DOCUMENT == | * Single particle tomography USER'S GUIDE (updated on June 2012; under major refactoring do to extensive changes in e2spt capabilities; look for an extensively different version soon) |
| Line 17: | Line 12: |
| [[attachment:eman2_spt_tutorial.pdf| eman2_spt_tutorial.pdf|&do=get]] | [[attachment:e2spt_users_guide_06_2012.pdf| e2spt_users_guide_06_2012.pdf|&do=get]] |
| Line 20: | Line 15: |
| Epsilon 15 virus data, used since the EMAN2 Workshop in 2010. | |
| Line 21: | Line 17: |
| [[attachment:e15tomograms.zip| e15tomograms.zip|&do=get]] Get all the tomograms listed above in a zip file |
[[http://blake.grid.bcm.edu/wikifiles/SPT/e2spt_data.zip|e2spt_data.zip]] |
| Line 24: | Line 19: |
| TRiC chapeornin data (NOT READY). | |
| Line 25: | Line 21: |
| . | [[attachment:e2spt_data_apoTRiC.zip| e2spt_data_apoTRiC.zip|&do=get]] |
| Line 27: | Line 23: |
| . | == COMMANDS (for easy "copy-pasting" into the command line == |
| Line 29: | Line 25: |
| . | === Monstrous command for alignment with e2spt_classaverage.py (used to be e2classaverage3d.py) === |
| Line 31: | Line 27: |
| . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . = ANYTHING BELOW THIS MARK IS DEPRECATED = This session will cover the beta version of a small fraction of the possibilities for SPT processing EMAN2 will eventually offer. = RESULTS UH = [[attachment:e15normal_average.hdf| e15nromal_average.hdf|&do=get]] [[attachment:e15normal_average_lp50.hdf| e15nromal_average_lp50.hdf|&do=get]] == OUTLINE == * 1) SPT processing through EMAN2's workflow (e2workflow.py) * 2) Sub-volume extraction from tomograms using EMAN2's 3D particle picking tool (e2tomoboxer.py) * 3) "Preparation" of extracted particles for alignment. [Details later. /!\ For a myriad of reasons, it is NOT recommendable to align and average sub-volumes directly after extraction without "preparing" them first]. * 4) Reference-based alignment and averaging. <!> ''PLEASE NOTE that "particle" and "sub-volume" are used interchangeably'' |
e2spt_classaverage.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 --lowpass=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --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:search=2 --averager=mean.tomo --aligncmp=ccc.tomo --raligncmp=ccc.tomo --shrink=3 --shrinkrefine=3 --savesteps --saveali --iter=8 --normproc=normalize --sym=c1 --keep=0.8 --path=whatever_folder_I_want |
| Line 131: | Line 31: |
| === Monstrous command for alignment with e2spt_hac.py (used to be e2tomoallvsall.py) === | |
| Line 132: | Line 33: |
| == BOXING == There are two options for opening the tomogram for purposes of boxing it. |
e2spt_hac.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 |
| Line 135: | Line 35: |
| 1) Directly, by typing e2tomoboxer.py followed my the path to the tomogram file at the commandline. | |
| Line 137: | Line 36: |
| 2) Or you can launch e2workflow.py from the commandline and access a tomogram through the browser in the tomographic menu. | === Not-so-monstrous command for e2spt_simulation.py (used to be e2tomosim.py) === e2spt_simulation.py --input=../groRef_scaled_bin2.hdf --snr=5 --nptcls=2 --tiltstep=5 --tiltrange=60 --transrange=10 --saveprjs --noiseproc=math.addnoise |
| Line 139: | Line 39: |
| [In theory, you can open a tomogram for contemplation purposes by typing: "e2display.py <my_tomogram_path_name_goes_here>" at the commandline. /!\ This is NOT recommendable, unless you have a grossly large (VERY, VERY large) amount of virtual memory on your computer; otherwise, catastrophe WILL befall upon you]. |
|
| Line 142: | Line 40: |
| Let's explore the FIRST APPROACH. | === Semi monstrous command for e2spt_resolutionplot.py === e2spt_resolutionplot.py --vol1=half1avg.hdf --vol2=half2avg.hdf --output=whatever3.txt --npeakstorefine=1 --verbose=3 --shrink=3 --shrinkrefine=2 --mask=mask.sharp:outer_radius=36 --preprocess=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --align=rotate_translate_3d:search=4:dphi=30:delta=30:sym=icos --parallel=thread:8 --ralign=refine_3d_grid:delta=15:range=30:search=2 --aligncmp=ccc.tomo --raligncmp=ccc.tomo --postprocess=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --normproc=normalize --sym=icos |
| Line 144: | Line 43: |
| === OPENING A TOMOGRAM DIRECTLY WITH e2tomoboxer.py === To launch the GUI that will show you the tomogram (you're free to choose whichever you prefer, "e15normal.rec" or "e15phaseplate.rec") type the following COMMAND #001 at the commandline: |
|
| Line 147: | Line 44: |
| ||<style="color: #FF0000; font-weight: bold;">COMMAND #001|| {{{ e2tomoboxer.py e15normal.rec --yshort --inmemory }}} |
=== Decent command for e2spt_rotationalplot.py === e2spt_rotationalplot.py --input=initModel.hdf --output=toAs129avsaAVG.txt --daz=1 --shrink=1 --dalt=180 --mask=mask.sharp:outer_radius=28 |
| Line 152: | Line 47: |
| ''' ''EXPLANATION'' ''' | |
| Line 154: | Line 48: |
| '''To specify --yshort, or not to''' This option will FLIP the orientation of your tomogram respect to the Y and Z coordinates. You should ONLY specify it IF the tomogram has its smallest/slimmest dimension running along the Y-axis, so that it becomes parallel to the Z-axis instead. '''Ok... but WHY? What does this mean?''' Some tomograms are built with the slimmest part of the 3D volume (that corresponding to the "ice thickness" in cryoEM) running along the Y-axis. BUT '''__in EMAN2 volumes are displayed such that the Z-axis is perpendicular to the screen__'''. If you open one of these tomograms "as is", you'll be looking at it "from the side". Most of the time you want the slimmest part of the volume to be aligned along Z (NOT Y), so you can see the tomogram "from the TOP", and look at the entire CCD-captured area in the XY plane slice by slice, as you go through the volume. '''--inmemory''' This option pre-loads your tomogram to memory, allowing to box particles (or do whatever you want to do in e2tomoboxer) more smoothly (faster), because reading data from memory is faster than reading it from disk. You can get the entire list of options that e2tomoboxer.py takes (not many at this point) and some sort of explanation of what they're for by typing the following at the commandline: {{{ e2tomoboxer.py -h }}} If you ran the command and took a leap of faith in skipping the explanation, you will probably have already clicked a few things in the GUI (Graphical User Interface with nice 'clickable' buttons) that popped up. ''~-(I hope you did; extensive explanations really ought to be read only when following the execution boxes doesn't work smoothly, when you're confused, when you have a lot of time to spare and nothing better to do, or when you're a purist [slightly OCD] about reading manuals in their entirety with the noble purpose of getting the most out of them).-~'' === USING THE WORKFLOW FOR SPT === The e2tomoboxer.py GUI should look like this: {{attachment:image.png|alt text|width=100 height=150}} This window is called __ You will notice... |
=== Command for e2spt_radialdensityplot.py === e2spt_radialdensityplot.py --vols=volA_aligned.hdf,volB_aligned.hdf --normproc=normalize.edgemean --lowpass=filter.lowpass.gauss:cutoff_freq=0.02:apix=4.401 --singleplot --output=volAali_VS_volBali.png |
Single Particle Tomography in EMAN2
Unfortunately, this technique SPT is very computationally intensive (both in terms of memory and processing speed). 3GB of RAM is the bare minimum recommended to get through the tutorial. For more realistic SPT on full 3D alignments on large sets of large particles particles, 8GB of memory and the use of multiple processing units is advised.
E2SPT USERS' GUIDE
* Single particle tomography USER'S GUIDE (updated on June 2012; under major refactoring do to extensive changes in e2spt capabilities; look for an extensively different version soon)
DATA
Epsilon 15 virus data, used since the EMAN2 Workshop in 2010.
TRiC chapeornin data (NOT READY).
COMMANDS (for easy "copy-pasting" into the command line
Monstrous command for alignment with e2spt_classaverage.py (used to be e2classaverage3d.py)
e2spt_classaverage.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 --lowpass=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --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:search=2 --averager=mean.tomo --aligncmp=ccc.tomo --raligncmp=ccc.tomo --shrink=3 --shrinkrefine=3 --savesteps --saveali --iter=8 --normproc=normalize --sym=c1 --keep=0.8 --path=whatever_folder_I_want
Monstrous command for alignment with e2spt_hac.py (used to be e2tomoallvsall.py)
e2spt_hac.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
Not-so-monstrous command for e2spt_simulation.py (used to be e2tomosim.py)
e2spt_simulation.py --input=../groRef_scaled_bin2.hdf --snr=5 --nptcls=2 --tiltstep=5 --tiltrange=60 --transrange=10 --saveprjs --noiseproc=math.addnoise
Semi monstrous command for e2spt_resolutionplot.py
e2spt_resolutionplot.py --vol1=half1avg.hdf --vol2=half2avg.hdf --output=whatever3.txt --npeakstorefine=1 --verbose=3 --shrink=3 --shrinkrefine=2 --mask=mask.sharp:outer_radius=36 --preprocess=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --align=rotate_translate_3d:search=4:dphi=30:delta=30:sym=icos --parallel=thread:8 --ralign=refine_3d_grid:delta=15:range=30:search=2 --aligncmp=ccc.tomo --raligncmp=ccc.tomo --postprocess=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --normproc=normalize --sym=icos
Decent command for e2spt_rotationalplot.py
e2spt_rotationalplot.py --input=initModel.hdf --output=toAs129avsaAVG.txt --daz=1 --shrink=1 --dalt=180 --mask=mask.sharp:outer_radius=28
Command for e2spt_radialdensityplot.py
e2spt_radialdensityplot.py --vols=volA_aligned.hdf,volB_aligned.hdf --normproc=normalize.edgemean --lowpass=filter.lowpass.gauss:cutoff_freq=0.02:apix=4.401 --singleplot --output=volAali_VS_volBali.png