EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

e2refine_easy.py report Note - This is a beta release version of EMAN2.1 This program is fully functional, but we will continue to add new features and refinements as we approach the final release. If you are curious to see a list of the exact refinement parameters used, browse to the 0_refine_parms.json file in the refinement directory. You can use 'Info' in the file browser or just read the file directly (.json files are plain text)

Explantion of Refinement Parameters Randomizing the Fourier phases of 1ss8.ali.2.10.mrc at resolutions higher than 12.0 Å. If the final achieved resolution is not at least ~10.8 Å, then the gold standard resolution assessment is not valid, and you need to re-refine, starting with a lower resolution target. Input particles are from sets/all__ctf_flip_hp.lst Several different methods can be used for final amplitude correction in cryoEM. The most accurate of these is to take the final structure and filter it so its 1-D power spectrum matches a known 1-D power spectrum from X-ray solution scattering or other source. This 'ideally filtered' structure is then low-pass filtered based on the same FSC curve used to measure resolution between the independent even/odd models used in the Gold Standard resolution criterion. This is the normal method EMAN2 will use for final corrections. Other choices are possible as well, however. EMAN2 generates an experimental structure factor directly from your data as part of the CTF fitting procedure. The low resolution portion is drawn directly from your data and the high resolution portion (generally past 14-18 A) is an empirical structure factor for proteins in general with a mix of alpha and beta components. This works quite well for most proteins, however is not completely appropriate for hybrid molecules with nucleotides (like ribosomes), but will generally still produce reasonable structures. Note that this is just a standard linear filter, which is not really changing the 3-D structure at all, it's really just changing how that structure is portrayed. The structure factor produced when CTF fitting is stored in strucfac.txt in the project directory. If you replace this file with a structure factor from some other source, it will be used to process all subsequent particles. Based on your selected --speed, I am setting --sep 3. This puts each particle into its 3 best orientations. If the angular sampling is finer than required to achieve the specified resolution, then a certain amount of rotational 'smearing' of each particle will help improve SNR in the resulting map without the 'smearing' degrading the actual map quality. This can achieve maximum liklihood-like effects without the substantial compuations this can entail. If you are concerned by this, or have many more particles than are really required to achieve the targeted resolution, you may consider manually specifiying --sep 1, which will override this automatic behavior. Your desired resolution is beyond 3/4 Nyquist. Regardless, we will set --classiter to 1 initially. Leaving this above 0 will help avoid noise bias, but it may be reduced to zero if convergence seems to have been achieved. The resolution you are requesting is beyond 2/3 Nyquist. This is normally not recommended, as it represents insufficient sampling to give a good representation of your reconstructed map, and resolution can be difficult to accurately assess. The reconstruction will proceed, but generally speaking your A/pix should be less than 1/3 the targeted resolution. Based on your requested resolution and box-size, modified by --speed, I will use an angular sampling of 3.60 deg. For details, please see http://blake.bcm.edu/emanwiki/EMAN2/AngStep As particles get translated during alignment, the total amount of noise present in the aligned

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05/27/2014 02:43 PM

EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

particle can change significantly. While this isn't a very large effect for similarity metrics like fsc, it can cause a bias in the reconstruction. Similarly if we use a different mask derived for each projection to combat this problem (as with the zeromask=1 option in some comparators, then each projection masks out a different fraction of the image causing some orientations to be preferred. To combat both effects, I will compute a single aggregate mask from all of the projections in each iteration, and use it as --mask refine_30/simmask.hdf. The mask is autogenerated and overwritten after each iteration. The only way to completely disable this behavior is to specify --simmask yourself with a file containing all 1.0 pixels.

Analysis of Refinement Results Convergence Analysis The plot below shows the FSC computed between iterations for both even and odd particle subsets. It is not a measure of resolution, but is a measure of how much the individual even/odd maps are changing from one iteration to the next. In a perfect world, this plot would eventually be 1.0 indicating no change from one iteration to the next after the refinement converges. In reality we normally expect only a pseudoconvergence, where the curves approach a final shape but do not actually become 1.0. This plot is automatically updated after each iteration.

Gold Standard Resolution

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EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

e2refine_easy computes a true gold-standard resolution as part of its stanard processing. This curve is also used as a basis for filtering the maps. Resolution can be gagued as the point at which this curve falls below a value of 0.143. Note that when computing FSCs in other situations, for example, when comparing the final map produced with all of the particle data to a higher resolution crystal structure, the more stringent 0.5 (actually 0.4) criterion must be used. Don't overinterpret these plots. The FSC plots themselves contain some noise, so there is some uncertainty in any resolution value.

Iteration 1: Resolution = 8.4 Å (gold standard refinement, FSC @0.143) Iteration 2: Resolution = 8.2 Å (gold standard refinement, FSC @0.143) Iteration 3: Resolution = 8.2 Å (gold standard refinement, FSC @0.143) Iteration 4: Resolution = 8.2 Å (gold standard refinement, FSC @0.143) Iteration 5: Resolution = 8.2 Å (gold standard refinement, FSC @0.143) Iteration 6: Resolution = 8.1 Å (gold standard refinement, FSC @0.143) Iteration 7: Resolution = 8.2 Å (gold standard refinement, FSC @0.143) Congratulations, your refinement is complete, and you have a gold standard resolution of 8.2 Å. If you wish to continue this refinement to further improve resolution, the most efficient approach is to

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EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

use the --startfrom refine_30 option rather than specifying --input and --model. When you use --startfrom, it will not re-randomize the phases. Since you have already achieved sufficient resolution to validate the gold-standard approach, continuing to extend this resolution is valid, and more efficient.

Explore your results Here are some useful output files to look at: Your final 3-D map from this run is refine_30/threed_07.hdf It may be useful to compare refine_30/classes_07.hdf to refine_30/projections_07.hdf. The images in these two files should match extremely well, other than more noise being present in the class-averages. Run e2eulerxplor.py to look at the distribution of particle orientations and interactively compare projections to class-averages. Click on a specific peak to see the projection and corresponding filtered class-average for any specific orientation. If you wish to make a single stack where you can look at all projections/averages side-by-side: e2proc2d.py refine_30/classes_07.hdf clsvsproj.hdf --interlv refine_30/projections_07.hdf It may also be worthwhile to compare refine_30/threed_07_even.hdf to refine_30/threed_07_odd.hdf to observe the differences responsible for the assessed resolution. The individual FSC curves are in 2-column text files called fsc_*.txt. Both masked and unmasked FSCs are computed The reconstruction produced after each iteration (threed_??.hdf) is masked and filtered as part of the reconstruction process. For the final completed iteration, the unmasked even and odd volumes are also retained: threed_even|odd_unmasked.hdf

Detailed command log Tue May 27 11:53:53 2014: e2proc3d.py 1ss8.ali.2.10.mrc refine_30/threed_00_even.hdf --process=filter.lowpass.randomphase:cutoff_freq=0.0833333333333 --apix=2.1 Tue May 27 11:53:53 2014: e2proc3d.py 1ss8.ali.2.10.mrc refine_30/threed_00_odd.hdf --process=filter.lowpass.randomphase:cutoff_freq=0.0833333333333 --apix=2.1 Beginning iteration 1 at Tue May 27 11:53:54 2014 * Generating 2-D projections of even/odd 3-D maps Tue May 27 11:53:54 2014: e2project3d.py refine_30/threed_00_even.hdf --outfile refine_30/projections_01_even.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16 Tue May 27 11:54:01 2014: e2project3d.py refine_30/threed_00_odd.hdf --outfile refine_30/projections_01_odd.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16 * Computing similarity of each particle to the set of projections using a hierarchical scheme. This will be the basis for classification. Tue May 27 11:54:08 2014: e2simmx2stage.py refine_30/projections_01_even.hdf sets/all__ctf_flip_hp_even.lst refine_30/simmx_01_even.hdf refine_30/proj_simmx_01_even.hdf refine_30/proj_stg1_01_even.hdf refine_30/simmx_stg1_01_even.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp

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EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 Tue May 27 12:02:33 2014: e2simmx2stage.py refine_30/projections_01_odd.hdf sets/all__ctf_flip_hp_odd.lst refine_30/simmx_01_odd.hdf refine_30/proj_simmx_01_odd.hdf refine_30/proj_stg1_01_odd.hdf refine_30/simmx_stg1_01_odd.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 * Based on the similarity values, put each particle in to 1 or more classes (depending on --sep) Tue May 27 12:10:59 2014: e2classify.py refine_30/simmx_01_even.hdf refine_30/classmx_01_even.hdf -f --sep 3 Tue May 27 12:11:08 2014: e2classify.py refine_30/simmx_01_odd.hdf refine_30/classmx_01_odd.hdf -f --sep 3 * Iteratively align and average all of the particles within each class, discarding the worst fraction Tue May 27 12:11:16 2014: e2classaverage.py --input sets/all__ctf_flip_hp_even.lst --classmx refine_30/classmx_01_even.hdf --storebad --output refine_30/classes_01_even.hdf --ref refine_30/projections_01_even.hdf --iter 1 -f --resultmx refine_30/cls_result_01_even.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 Tue May 27 12:12:46 2014: e2classaverage.py --input sets/all__ctf_flip_hp_odd.lst --classmx refine_30/classmx_01_odd.hdf --storebad --output refine_30/classes_01_odd.hdf --ref refine_30/projections_01_odd.hdf --iter 1 -f --resultmx refine_30/cls_result_01_odd.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 * Using the known orientations, reconstruct the even/odd 3-D maps from the even/odd 2-D classaverages. Tue May 27 12:14:18 2014: e2make3dpar.py --input refine_30/classes_01_even.hdf --sym d7 --output refine_30/threed_01_even.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt Tue May 27 12:14:28 2014: e2make3dpar.py --input refine_30/classes_01_odd.hdf --sym d7 --output refine_30/threed_01_odd.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt * Finally, determine the resolution, filter and mask the even/odd maps, and then produce the final 3-D map for this iteration. Note that the next iteration is seeded with the individual even/odd maps, not the final average. Tue May 27 12:14:38 2014: e2refine_postprocess.py --even refine_30/threed_01_even.hdf --odd refine_30/threed_01_odd.hdf --output refine_30/threed_01.hdf --align --mass 800.0 --iter 1 --sym=d7 --underfilter Tue May 27 12:14:44 2014: e2proc3d.py refine_30/threed_01_even.hdf refine_30/converge_even_00_01.txt --calcfsc refine_30/threed_00_even.hdf Tue May 27 12:14:44 2014: e2proc3d.py refine_30/threed_01_odd.hdf refine_30/converge_odd_00_01.txt --calcfsc refine_30/threed_00_odd.hdf

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05/27/2014 02:43 PM

EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

Beginning iteration 2 at Tue May 27 12:14:45 2014 * Generating 2-D projections of even/odd 3-D maps Tue May 27 12:14:45 2014: e2project3d.py refine_30/threed_01_even.hdf --outfile refine_30/projections_02_even.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16 Tue May 27 12:14:52 2014: e2project3d.py refine_30/threed_01_odd.hdf --outfile refine_30/projections_02_odd.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16 * Computing similarity of each particle to the set of projections using a hierarchical scheme. This will be the basis for classification. Tue May 27 12:14:59 2014: e2simmx2stage.py refine_30/projections_02_even.hdf sets/all__ctf_flip_hp_even.lst refine_30/simmx_02_even.hdf refine_30/proj_simmx_02_even.hdf refine_30/proj_stg1_02_even.hdf refine_30/simmx_stg1_02_even.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 Tue May 27 12:23:23 2014: e2simmx2stage.py refine_30/projections_02_odd.hdf sets/all__ctf_flip_hp_odd.lst refine_30/simmx_02_odd.hdf refine_30/proj_simmx_02_odd.hdf refine_30/proj_stg1_02_odd.hdf refine_30/simmx_stg1_02_odd.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 * Based on the similarity values, put each particle in to 1 or more classes (depending on --sep) Tue May 27 12:31:58 2014: e2classify.py refine_30/simmx_02_even.hdf refine_30/classmx_02_even.hdf -f --sep 3 Tue May 27 12:32:06 2014: e2classify.py refine_30/simmx_02_odd.hdf refine_30/classmx_02_odd.hdf -f --sep 3 * Iteratively align and average all of the particles within each class, discarding the worst fraction Tue May 27 12:32:16 2014: e2classaverage.py --input sets/all__ctf_flip_hp_even.lst --classmx refine_30/classmx_02_even.hdf --storebad --output refine_30/classes_02_even.hdf --ref refine_30/projections_02_even.hdf --iter 1 -f --resultmx refine_30/cls_result_02_even.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 Tue May 27 12:33:48 2014: e2classaverage.py --input sets/all__ctf_flip_hp_odd.lst --classmx refine_30/classmx_02_odd.hdf --storebad --output refine_30/classes_02_odd.hdf --ref refine_30/projections_02_odd.hdf --iter 1 -f --resultmx refine_30/cls_result_02_odd.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 * Using the known orientations, reconstruct the even/odd 3-D maps from the even/odd 2-D classaverages. Tue May 27 12:35:21 2014: e2make3dpar.py --input refine_30/classes_02_even.hdf --sym d7 --output

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EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

refine_30/threed_02_even.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt Tue May 27 12:35:31 2014: e2make3dpar.py --input refine_30/classes_02_odd.hdf --sym d7 --output refine_30/threed_02_odd.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt * Finally, determine the resolution, filter and mask the even/odd maps, and then produce the final 3-D map for this iteration. Note that the next iteration is seeded with the individual even/odd maps, not the final average. Tue May 27 12:35:41 2014: e2refine_postprocess.py --even refine_30/threed_02_even.hdf --odd refine_30/threed_02_odd.hdf --output refine_30/threed_02.hdf --align --mass 800.0 --iter 2 --sym=d7 --underfilter Tue May 27 12:35:47 2014: e2proc3d.py refine_30/threed_02_even.hdf refine_30/converge_even_01_02.txt --calcfsc refine_30/threed_01_even.hdf Tue May 27 12:35:47 2014: e2proc3d.py refine_30/threed_02_odd.hdf refine_30/converge_odd_01_02.txt --calcfsc refine_30/threed_01_odd.hdf Beginning iteration 3 at Tue May 27 12:35:48 2014 * Generating 2-D projections of even/odd 3-D maps Tue May 27 12:35:48 2014: e2project3d.py refine_30/threed_02_even.hdf --outfile refine_30/projections_03_even.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16 Tue May 27 12:35:55 2014: e2project3d.py refine_30/threed_02_odd.hdf --outfile refine_30/projections_03_odd.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16 * Computing similarity of each particle to the set of projections using a hierarchical scheme. This will be the basis for classification. Tue May 27 12:36:02 2014: e2simmx2stage.py refine_30/projections_03_even.hdf sets/all__ctf_flip_hp_even.lst refine_30/simmx_03_even.hdf refine_30/proj_simmx_03_even.hdf refine_30/proj_stg1_03_even.hdf refine_30/simmx_stg1_03_even.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 Tue May 27 12:44:36 2014: e2simmx2stage.py refine_30/projections_03_odd.hdf sets/all__ctf_flip_hp_odd.lst refine_30/simmx_03_odd.hdf refine_30/proj_simmx_03_odd.hdf refine_30/proj_stg1_03_odd.hdf refine_30/simmx_stg1_03_odd.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 * Based on the similarity values, put each particle in to 1 or more classes (depending on --sep) Tue May 27 12:53:21 2014: e2classify.py refine_30/simmx_03_even.hdf refine_30/classmx_03_even.hdf -f --sep 3 Tue May 27 12:53:29 2014: e2classify.py refine_30/simmx_03_odd.hdf refine_30/classmx_03_odd.hdf -f --sep 3

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EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

* Iteratively align and average all of the particles within each class, discarding the worst fraction Tue May 27 12:53:39 2014: e2classaverage.py --input sets/all__ctf_flip_hp_even.lst --classmx refine_30/classmx_03_even.hdf --storebad --output refine_30/classes_03_even.hdf --ref refine_30/projections_03_even.hdf --iter 1 -f --resultmx refine_30/cls_result_03_even.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 Tue May 27 12:55:12 2014: e2classaverage.py --input sets/all__ctf_flip_hp_odd.lst --classmx refine_30/classmx_03_odd.hdf --storebad --output refine_30/classes_03_odd.hdf --ref refine_30/projections_03_odd.hdf --iter 1 -f --resultmx refine_30/cls_result_03_odd.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 * Using the known orientations, reconstruct the even/odd 3-D maps from the even/odd 2-D classaverages. Tue May 27 12:56:41 2014: e2make3dpar.py --input refine_30/classes_03_even.hdf --sym d7 --output refine_30/threed_03_even.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt Tue May 27 12:56:51 2014: e2make3dpar.py --input refine_30/classes_03_odd.hdf --sym d7 --output refine_30/threed_03_odd.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt * Finally, determine the resolution, filter and mask the even/odd maps, and then produce the final 3-D map for this iteration. Note that the next iteration is seeded with the individual even/odd maps, not the final average. Tue May 27 12:57:01 2014: e2refine_postprocess.py --even refine_30/threed_03_even.hdf --odd refine_30/threed_03_odd.hdf --output refine_30/threed_03.hdf --align --mass 800.0 --iter 3 --sym=d7 --underfilter Tue May 27 12:57:07 2014: e2proc3d.py refine_30/threed_03_even.hdf refine_30/converge_even_02_03.txt --calcfsc refine_30/threed_02_even.hdf Tue May 27 12:57:07 2014: e2proc3d.py refine_30/threed_03_odd.hdf refine_30/converge_odd_02_03.txt --calcfsc refine_30/threed_02_odd.hdf Beginning iteration 4 at Tue May 27 12:57:08 2014 * Generating 2-D projections of even/odd 3-D maps Tue May 27 12:57:08 2014: e2project3d.py refine_30/threed_03_even.hdf --outfile refine_30/projections_04_even.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16 Tue May 27 12:57:15 2014: e2project3d.py refine_30/threed_03_odd.hdf --outfile refine_30/projections_04_odd.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16 * Computing similarity of each particle to the set of projections using a hierarchical scheme. This will be the basis for classification.

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EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

Tue May 27 12:57:22 2014: e2simmx2stage.py refine_30/projections_04_even.hdf sets/all__ctf_flip_hp_even.lst refine_30/simmx_04_even.hdf refine_30/proj_simmx_04_even.hdf refine_30/proj_stg1_04_even.hdf refine_30/simmx_stg1_04_even.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 Tue May 27 13:06:07 2014: e2simmx2stage.py refine_30/projections_04_odd.hdf sets/all__ctf_flip_hp_odd.lst refine_30/simmx_04_odd.hdf refine_30/proj_simmx_04_odd.hdf refine_30/proj_stg1_04_odd.hdf refine_30/simmx_stg1_04_odd.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 * Based on the similarity values, put each particle in to 1 or more classes (depending on --sep) Tue May 27 13:14:32 2014: e2classify.py refine_30/simmx_04_even.hdf refine_30/classmx_04_even.hdf -f --sep 3 Tue May 27 13:14:41 2014: e2classify.py refine_30/simmx_04_odd.hdf refine_30/classmx_04_odd.hdf -f --sep 3 * Iteratively align and average all of the particles within each class, discarding the worst fraction Tue May 27 13:14:50 2014: e2classaverage.py --input sets/all__ctf_flip_hp_even.lst --classmx refine_30/classmx_04_even.hdf --storebad --output refine_30/classes_04_even.hdf --ref refine_30/projections_04_even.hdf --iter 1 -f --resultmx refine_30/cls_result_04_even.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 Tue May 27 13:16:20 2014: e2classaverage.py --input sets/all__ctf_flip_hp_odd.lst --classmx refine_30/classmx_04_odd.hdf --storebad --output refine_30/classes_04_odd.hdf --ref refine_30/projections_04_odd.hdf --iter 1 -f --resultmx refine_30/cls_result_04_odd.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 * Using the known orientations, reconstruct the even/odd 3-D maps from the even/odd 2-D classaverages. Tue May 27 13:17:55 2014: e2make3dpar.py --input refine_30/classes_04_even.hdf --sym d7 --output refine_30/threed_04_even.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt Tue May 27 13:18:05 2014: e2make3dpar.py --input refine_30/classes_04_odd.hdf --sym d7 --output refine_30/threed_04_odd.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt * Finally, determine the resolution, filter and mask the even/odd maps, and then produce the final 3-D map for this iteration. Note that the next iteration is seeded with the individual even/odd maps, not the final average. Tue May 27 13:18:17 2014: e2refine_postprocess.py --even refine_30/threed_04_even.hdf --odd refine_30/threed_04_odd.hdf --output refine_30/threed_04.hdf --align --mass 800.0 --iter 4 --sym=d7 --underfilter Tue May 27 13:18:22 2014: e2proc3d.py refine_30/threed_04_even.hdf refine_30/converge_even_03_04.txt --calcfsc refine_30/threed_03_even.hdf

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05/27/2014 02:43 PM

EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

Tue May 27 13:18:22 2014: e2proc3d.py refine_30/threed_04_odd.hdf refine_30/converge_odd_03_04.txt --calcfsc refine_30/threed_03_odd.hdf Beginning iteration 5 at Tue May 27 13:18:23 2014 * Generating 2-D projections of even/odd 3-D maps Tue May 27 13:18:23 2014: e2project3d.py refine_30/threed_04_even.hdf --outfile refine_30/projections_05_even.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16 Tue May 27 13:18:30 2014: e2project3d.py refine_30/threed_04_odd.hdf --outfile refine_30/projections_05_odd.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16 * Computing similarity of each particle to the set of projections using a hierarchical scheme. This will be the basis for classification. Tue May 27 13:18:37 2014: e2simmx2stage.py refine_30/projections_05_even.hdf sets/all__ctf_flip_hp_even.lst refine_30/simmx_05_even.hdf refine_30/proj_simmx_05_even.hdf refine_30/proj_stg1_05_even.hdf refine_30/simmx_stg1_05_even.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 Tue May 27 13:27:01 2014: e2simmx2stage.py refine_30/projections_05_odd.hdf sets/all__ctf_flip_hp_odd.lst refine_30/simmx_05_odd.hdf refine_30/proj_simmx_05_odd.hdf refine_30/proj_stg1_05_odd.hdf refine_30/simmx_stg1_05_odd.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 * Based on the similarity values, put each particle in to 1 or more classes (depending on --sep) Tue May 27 13:35:26 2014: e2classify.py refine_30/simmx_05_even.hdf refine_30/classmx_05_even.hdf -f --sep 3 Tue May 27 13:35:35 2014: e2classify.py refine_30/simmx_05_odd.hdf refine_30/classmx_05_odd.hdf -f --sep 3 * Iteratively align and average all of the particles within each class, discarding the worst fraction Tue May 27 13:35:44 2014: e2classaverage.py --input sets/all__ctf_flip_hp_even.lst --classmx refine_30/classmx_05_even.hdf --storebad --output refine_30/classes_05_even.hdf --ref refine_30/projections_05_even.hdf --iter 1 -f --resultmx refine_30/cls_result_05_even.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 Tue May 27 13:37:13 2014: e2classaverage.py --input sets/all__ctf_flip_hp_odd.lst --classmx refine_30/classmx_05_odd.hdf --storebad --output refine_30/classes_05_odd.hdf --ref refine_30/projections_05_odd.hdf --iter 1 -f --resultmx refine_30/cls_result_05_odd.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 * Using the known orientations, reconstruct the even/odd 3-D maps from the even/odd 2-D class-

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05/27/2014 02:43 PM

EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

averages. Tue May 27 13:38:43 2014: e2make3dpar.py --input refine_30/classes_05_even.hdf --sym d7 --output refine_30/threed_05_even.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt Tue May 27 13:38:53 2014: e2make3dpar.py --input refine_30/classes_05_odd.hdf --sym d7 --output refine_30/threed_05_odd.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt * Finally, determine the resolution, filter and mask the even/odd maps, and then produce the final 3-D map for this iteration. Note that the next iteration is seeded with the individual even/odd maps, not the final average. Tue May 27 13:39:03 2014: e2refine_postprocess.py --even refine_30/threed_05_even.hdf --odd refine_30/threed_05_odd.hdf --output refine_30/threed_05.hdf --align --mass 800.0 --iter 5 --sym=d7 --underfilter Tue May 27 13:39:08 2014: e2proc3d.py refine_30/threed_05_even.hdf refine_30/converge_even_04_05.txt --calcfsc refine_30/threed_04_even.hdf Tue May 27 13:39:09 2014: e2proc3d.py refine_30/threed_05_odd.hdf refine_30/converge_odd_04_05.txt --calcfsc refine_30/threed_04_odd.hdf Beginning iteration 6 at Tue May 27 13:39:09 2014 * Generating 2-D projections of even/odd 3-D maps Tue May 27 13:39:09 2014: e2project3d.py refine_30/threed_05_even.hdf --outfile refine_30/projections_06_even.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16 Tue May 27 13:39:16 2014: e2project3d.py refine_30/threed_05_odd.hdf --outfile refine_30/projections_06_odd.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16 * Computing similarity of each particle to the set of projections using a hierarchical scheme. This will be the basis for classification. Tue May 27 13:39:23 2014: e2simmx2stage.py refine_30/projections_06_even.hdf sets/all__ctf_flip_hp_even.lst refine_30/simmx_06_even.hdf refine_30/proj_simmx_06_even.hdf refine_30/proj_stg1_06_even.hdf refine_30/simmx_stg1_06_even.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 Tue May 27 13:47:58 2014: e2simmx2stage.py refine_30/projections_06_odd.hdf sets/all__ctf_flip_hp_odd.lst refine_30/simmx_06_odd.hdf refine_30/proj_simmx_06_odd.hdf refine_30/proj_stg1_06_odd.hdf refine_30/simmx_stg1_06_odd.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 * Based on the similarity values, put each particle in to 1 or more classes (depending on --sep) Tue May 27 13:56:22 2014: e2classify.py refine_30/simmx_06_even.hdf refine_30/classmx_06_even.hdf -f --sep 3

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05/27/2014 02:43 PM

EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

Tue May 27 13:56:31 2014: e2classify.py refine_30/simmx_06_odd.hdf refine_30/classmx_06_odd.hdf -f --sep 3 * Iteratively align and average all of the particles within each class, discarding the worst fraction Tue May 27 13:56:40 2014: e2classaverage.py --input sets/all__ctf_flip_hp_even.lst --classmx refine_30/classmx_06_even.hdf --storebad --output refine_30/classes_06_even.hdf --ref refine_30/projections_06_even.hdf --iter 1 -f --resultmx refine_30/cls_result_06_even.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 Tue May 27 13:58:12 2014: e2classaverage.py --input sets/all__ctf_flip_hp_odd.lst --classmx refine_30/classmx_06_odd.hdf --storebad --output refine_30/classes_06_odd.hdf --ref refine_30/projections_06_odd.hdf --iter 1 -f --resultmx refine_30/cls_result_06_odd.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 * Using the known orientations, reconstruct the even/odd 3-D maps from the even/odd 2-D classaverages. Tue May 27 13:59:45 2014: e2make3dpar.py --input refine_30/classes_06_even.hdf --sym d7 --output refine_30/threed_06_even.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt Tue May 27 13:59:55 2014: e2make3dpar.py --input refine_30/classes_06_odd.hdf --sym d7 --output refine_30/threed_06_odd.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt * Finally, determine the resolution, filter and mask the even/odd maps, and then produce the final 3-D map for this iteration. Note that the next iteration is seeded with the individual even/odd maps, not the final average. Tue May 27 14:00:05 2014: e2refine_postprocess.py --even refine_30/threed_06_even.hdf --odd refine_30/threed_06_odd.hdf --output refine_30/threed_06.hdf --align --mass 800.0 --iter 6 --sym=d7 --underfilter Tue May 27 14:00:10 2014: e2proc3d.py refine_30/threed_06_even.hdf refine_30/converge_even_05_06.txt --calcfsc refine_30/threed_05_even.hdf Tue May 27 14:00:11 2014: e2proc3d.py refine_30/threed_06_odd.hdf refine_30/converge_odd_05_06.txt --calcfsc refine_30/threed_05_odd.hdf Beginning iteration 7 at Tue May 27 14:00:12 2014 * Generating 2-D projections of even/odd 3-D maps Tue May 27 14:00:12 2014: e2project3d.py refine_30/threed_06_even.hdf --outfile refine_30/projections_07_even.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16 Tue May 27 14:00:19 2014: e2project3d.py refine_30/threed_06_odd.hdf --outfile refine_30/projections_07_odd.hdf -f --projector standard --orientgen eman:delta=3.59960:inc_mirror=0:perturb=0 --sym d7 --postprocess normalize.circlemean --parallel thread:16

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05/27/2014 02:43 PM

EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

* Computing similarity of each particle to the set of projections using a hierarchical scheme. This will be the basis for classification. Tue May 27 14:00:26 2014: e2simmx2stage.py refine_30/projections_07_even.hdf sets/all__ctf_flip_hp_even.lst refine_30/simmx_07_even.hdf refine_30/proj_simmx_07_even.hdf refine_30/proj_stg1_07_even.hdf refine_30/simmx_stg1_07_even.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 Tue May 27 14:09:00 2014: e2simmx2stage.py refine_30/projections_07_odd.hdf sets/all__ctf_flip_hp_odd.lst refine_30/simmx_07_odd.hdf refine_30/proj_simmx_07_odd.hdf refine_30/proj_stg1_07_odd.hdf refine_30/simmx_stg1_07_odd.hdf --saveali --cmp frc:snrweight=1:maxres=7.0 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16 * Based on the similarity values, put each particle in to 1 or more classes (depending on --sep) Tue May 27 14:17:15 2014: e2classify.py refine_30/simmx_07_even.hdf refine_30/classmx_07_even.hdf -f --sep 3 Tue May 27 14:17:24 2014: e2classify.py refine_30/simmx_07_odd.hdf refine_30/classmx_07_odd.hdf -f --sep 3 * Iteratively align and average all of the particles within each class, discarding the worst fraction Tue May 27 14:17:33 2014: e2classaverage.py --input sets/all__ctf_flip_hp_even.lst --classmx refine_30/classmx_07_even.hdf --storebad --output refine_30/classes_07_even.hdf --ref refine_30/projections_07_even.hdf --iter 1 -f --resultmx refine_30/cls_result_07_even.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 Tue May 27 14:19:06 2014: e2classaverage.py --input sets/all__ctf_flip_hp_odd.lst --classmx refine_30/classmx_07_odd.hdf --storebad --output refine_30/classes_07_odd.hdf --ref refine_30/projections_07_odd.hdf --iter 1 -f --resultmx refine_30/cls_result_07_odd.hdf --normproc normalize.edgemean --averager ctfw.auto --keep 0.9 --cmp frc:snrweight=1 --align rotate_translate_flip --aligncmp ccc --ralign refine --raligncmp frc:snrweight=1:zeromask=1 --parallel thread:16 * Using the known orientations, reconstruct the even/odd 3-D maps from the even/odd 2-D classaverages. Tue May 27 14:20:35 2014: e2make3dpar.py --input refine_30/classes_07_even.hdf --sym d7 --output refine_30/threed_07_even.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt Tue May 27 14:20:46 2014: e2make3dpar.py --input refine_30/classes_07_odd.hdf --sym d7 --output refine_30/threed_07_odd.hdf --keep 0.5 --apix 2.1 --pad 180 --fillangle 3.5996 --threads 16 --setsf strucfac.txt * Finally, determine the resolution, filter and mask the even/odd maps, and then produce the final 3-D map for this iteration. Note that the next iteration is seeded with the individual even/odd maps, not the final average. Tue May 27 14:20:55 2014: e2refine_postprocess.py --even refine_30/threed_07_even.hdf --odd refine_30/threed_07_odd.hdf --output refine_30/threed_07.hdf --align --mass 800.0 --iter 7 --sym=d7 --underfilter

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05/27/2014 02:43 PM

EMAN2 Refinement Overview

file:///home/stevel/refine/workshop_beijing/refine...

Tue May 27 14:21:01 2014: e2proc3d.py refine_30/threed_07_even.hdf refine_30/converge_even_06_07.txt --calcfsc refine_30/threed_06_even.hdf Tue May 27 14:21:01 2014: e2proc3d.py refine_30/threed_07_odd.hdf refine_30/converge_odd_06_07.txt --calcfsc refine_30/threed_06_odd.hdf

Generated by EMAN 2.1 alpha3 $Date: 2014/05/26 17:01:06 $

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05/27/2014 02:43 PM

e2refine_easy.py report -

May 27, 2014 - frc:zeromask=1:snrweight=1 --shrinks1 2 --mask refine_30/simmask.hdf --parallel thread:16. Tue May 27 12:02:33 2014: e2simmx2stage.py ...

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