|
Size: 5418
Comment:
|
Size: 3356
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) |
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 9: | Line 7: |
| b) 3 e15 "references" (an asymmetric one, and one with icosahedral symmetry, both prepared for alignment; and a raw one, straight from the EMDB) | == DATA == Epsilon 15 virus data, used since the EMAN2 Workshop in 2010. [[http://blake.grid.bcm.edu/wikifiles/SPT/e2spt_data.zip|e2spt_data.zip]] |
| Line 11: | Line 11: |
| c) 1 text file (a small "coordinates file") Download the written tutorial provided through the PDF file below. [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]. |
TRiC chapeornin data (NOT READY). [[attachment:e2spt_data_apoTRiC.zip| e2spt_data_apoTRiC.zip|&do=get]] |
| Line 18: | Line 15: |
| Not available here for now. Get it through this site: | |
| Line 19: | Line 17: |
| [[attachment:eman2_spt_tutorial.pdf| eman2_spt_tutorial.pdf|&do=get]] | http://blake.bcm.edu/emanwiki/Ws2011/Agenda |
| Line 21: | Line 19: |
| == DATA == | == Monstrous command for alignment with e2spt_classaverage.py (used to be e2classaverage3d.py) == |
| Line 23: | Line 21: |
| [[attachment:e2spt_data.zip| e2spt_data.zip|&do=get]] Get all the tomograms listed above in a zip file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . = 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 --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: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 133: | Line 25: |
| == Monstrous command for alignment with e2spt_hac.py (used to be e2tomoallvsall.py) == | |
| Line 134: | Line 27: |
| == 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 137: | Line 29: |
| 1) Directly, by typing e2tomoboxer.py followed my the path to the tomogram file at the commandline. | |
| Line 139: | Line 30: |
| 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 141: | Line 33: |
| [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 144: | Line 34: |
| 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 146: | Line 37: |
| === 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 149: | Line 38: |
| ||<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 154: | Line 41: |
| ''' ''EXPLANATION'' ''' | |
| Line 156: | Line 42: |
| '''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.
DATA
Epsilon 15 virus data, used since the EMAN2 Workshop in 2010. e2spt_data.zip
TRiC chapeornin data (NOT READY). e2spt_data_apoTRiC.zip
TUTORIAL DOCUMENT
Not available here for now. Get it through this site:
http://blake.bcm.edu/emanwiki/Ws2011/Agenda
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 --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: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