Last update: October 1, 2013.

Before attempting to average subtomograms with EMAN2, make sure to have the latest EMAN2 release. Download the easy-to-install binaries for the "Daily Release" from EMAN2's download page:

http://ncmi.bcm.tmc.edu/ncmi/software/software_details?selected_software=counter_222

(The daily release is close to the middle of the page).

Single Particle Tomography in EMAN2

Unfortunately, SPT is very computationally intensive, both in terms of memory and processing speed. However, illustrative exercises and tutorials can be carried out on a laptop with 2-4GB of RAM. For more realistic SPT on full 3D alignments of large sets (hundreds of particles) comprising large particles (like viruses), 8GB of memory and the use of multiple processing units or GPU technology are advised.

E2SPT USERS' GUIDE

* Single particle tomography USER'S GUIDE (updated summer 2012; under major refactoring due to extensive changes in e2spt capabilities; look for a new and longer version of the tutorial soon).

e2spt_users_guide_june_2014_alpha.pdf

DATA

Epsilon 15 virus test data, used in the EMAN2 Workshop in 2011.

e2spt_data.zip

TRiC chapeornin test data (coming soon).

e2spt_data_apoTRiC.zip

Note that the EMAN2 Project Manager, launched through e2projectmanager.py, provides a full pipeline for SPT (particle selection, preprocessing, alignment and averaging) via iterative refinement. However, other additional alignment, processing and analysis tools are available from the command line.

COMMANDS (for easy "copy-pasting" into the command line; to see the entire list of parameters for each program, type the program name followed by -h at the command line)

CORE PROGRAMS

SPT BOXER

e2spt_boxer.py <tomogram.rec> --yshort --inmemory --lowpass=100

.

SPT Iterative refinement

Command for alignment with e2spt_classaverage.py (used to be e2classaverage3d.py)

e2spt_classaverage.py --input= --output= --ref= --npeakstorefine=14 -v 0 --mask=mask.sharp:outer_radius= --lowpass=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --parallel=thread:2 --highpass=filter.highpass.gauss:cutoff_freq=0.002:apix=4.401 --averager=mean.tomo --aligncmp=ccc.tomo --faligncmp=ccc.tomo --shrink=3 --shrinkfine=2 --savesteps --saveali --iter=24 --normproc=normalize --keep=0.8 --path= --align=rotate_translate_3d:search=10:delta=8:dphi=8:verbose=0:sym=icos --falign=refine_3d_grid:delta=3:range=9:search=2

.

SPT "all vs all"

Command for alignment with e2spt_hac.py (used to be e2tomoallvsall.py)

e2spt_hac.py -v 0 --path=AVSA --input= --shrink=3 --shrinkfine=2 --iter= --mask=mask.sharp:outer_radius= --npeakstorefine= --lowpass=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --highpass=filter.highpass.gauss:cutoff_freq=0.002:apix=4.401 --parallel=thread:24 --averager=mean.tomo --aligncmp=ccc.tomo --faligncmp=ccc.tomo --saveali --savesteps --postprocess=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --autocenter --plotccc --exclusive_class_min=8 --normproc=normalize --align=rotate_translate_3d:search=4:dphi=12:delta=12 --falign=refine_3d_grid:delta=3:range=12:search=2

.

OTHER programs (use at your own risk; some parameters in these programs are under experimental development. Tun the program at the command line followed by -h to see current available parameters for each program)

Command for e2spt_resolutionplot.py

e2spt_resolutionplot.py --vol1=half1avg.hdf --vol2=half2avg.hdf --output=whatever3.txt --npeakstorefine=1 --verbose=0 --shrink=3 --shrinkfine=2 --mask=mask.sharp:outer_radius=36 --lowpass=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --align=rotate_translate_3d:search=4:dphi=30:delta=30:sym=icos --parallel=thread:8 --falign=refine_3d_grid:delta=15:range=30:search=2 --aligncmp=ccc.tomo --faligncmp=ccc.tomo --normproc=normalize --sym=icos

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

Command for e2spt_simulation.py (used to be e2tomosim.py)

e2spt_simulation.py --input=groel.pdb --snr=5 --nptcls=8 --tiltstep=5 --tiltrange=60 --transrange=10 --saveprjs --addnoise --simref --path=TESTsimREF --pad=3 --shrink=2 --finalboxsize=96 --negativecontrast

Command for e2spt_tomosimjobs.py

e2spt_tomosimjobs.py --input=groel.pdb --nptcls=8 --saveprjs --addnoise --simref --path=TESTsimREF --pad=3 --shrink=2 --finalboxsize=96 --snrlowerlimit=0 --snrupperlimit=1 --snrchange=1 --tiltsteplowerlimit=0 --tiltstepupperlimit=1 --tiltstepchange=1 --tiltrangelowerlimit=60 --tiltrangeupperlimit=61 --tiltrangechange=1 --negativecontrast --testalignment

Command for e2spt_autoboxer.py

e2spt_autoboxer.py --tomogram=tomo_inv.rec --ptclradius=8 --path=whatever --concentrationfactor=1 --output=subtomostack.hdf --outputboxsize=36 --verbose=10 --goldstack=gold_ptcls_s05_inv.hdf --pruneprj --goldthreshtomo --keepn=150 --lowpass=filter.lowpass.gauss:cutoff_freq=0.02

Command for e2symsearch3d.py

e2symsearch3d.py --input= --output= --mask=

Command for e2spt_refinemulti.py

e2spt_refinemulti.py -v 0 --path=RF --input= --nrefs=2 --refgenmethod=binarytree --shrink=3 --shrinkfine=2 --iter= --mask=mask.sharp:outer_radius= --npeakstorefine= --lowpass=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --highpass=filter.highpass.gauss:cutoff_freq=0.002:apix=4.401 --parallel=thread:24 --averager=mean.tomo --aligncmp=ccc.tomo --faligncmp=ccc.tomo --saveali --savesteps --normproc=normalize --radius=150

Command for e2spt_fftamps.py

e2spt_fftamps.py --input=file.hdf

Command for e2spt_wedge.py (GUI available)