Building a preliminary model. There are specific instructions for generating a 'good' model for various symmetry possibilities. However, note that our current recommended technique is actually to start with a very bad answer, let the refinement converge, start with a different bad answer, refine to convergence again, and compare the results. If you can get the same results from 2-3 different starting models, you can be fairly confident that you have found the best structure. If you get different results in the trials, you can try 5-10 trials and see if one solution is obviously more correct (higher resolution, better agreement between projections, class averages and data). To generate a 'bad' starting model, ignore the instructions in this step and just run 'makeinitialmodel.py'. However make SURE that when you go to the 'refine' command in step 3 that you use the actual symmetry of your object. $csym
If you know your model has a Cn symmetry (n>2), or a Cn pseudosymmetry, it is generally very easy to generate an adequate preliminary model in EMAN. The command startcsym performs this task automatically from start.hed/img. This program will automatically search for particles which are top views (along the Cn axis) and side views of the macromolecule. It then generates a class average for both of these views, then builds a rough 3d model from the 2 class averages. This model will be VERY rough and noisy. This isn't generally a problem. In most cases this model will be sufficient for the refinement loop to converge. Note that it is critical that the particles you run startcsym on have been processed by cenalignint as described in step 1. You might also consider low-pass filtering the particles you are using for this step, using 'proc2d' with the 'lp=' and 'apix=' options. When you go on to the next step (iterative refinement), you can go back to the original start.hed which has not been centered. To use the program, type:
startcsym start.hed #-ptcl-to-use sym=cn
Replace n with the symmetry of your particle, eg 'c5' '#-ptcl-to-use' is the number of particles used to generate the top and side views. This number should generally be between 20 and 100, and should not be more than 5-10% of your total number of particles. A larger number will give less noisy class-averages, but they will be blurred out by averaging a wider range of different orientations. A smaller number will give a sharper but noisier result. If you have a LOT of particles (>5000), you might want to use only the first few thousand for this step to save time, eg:
proc2d start.hed tmp.hed last=3999 startcsym tmp.hed 60 sym=c4 rm tmp.hed tmp.img
After running this command, several new files will exist:
cls000.hed - the individual particles used to generate the top view
cls001.hed - the individual particles used to generate the side view
classes.hed - contains 2 class averages, the top view and the side view
sym.hed - contains 3 images, the symmetrized top view, the side view, and
the side view aligned to the symmetric axis
threed.0a.mrc - The preliminary 3d model.
You should take a look at classes.hed and sym.hed (use the eman file/history browser). Make sure that the symmetrized and unsymmetrized top views look somewhat similar. Make sure the side view isn't too ridiculous. If these are ok, chances are, the 3d model is good enough for the next step. Don't bother looking at the 3D model in projection. It will look terrible from most angles. An isosurface rendering will give you a better idea.
There is a possibility that the orientation of the side view may be determined incorrectly (the 3rd image in sym.img). Usually, if startcsym is wrong, it will be off by exactly 90 degrees. If this occurs, run:
startcsym start.hed #-ptcl-to-use sym=cn nosym fixrot=90
This will fix the 3D model withour rerunning the symmetry search. Double-check that it worked properly if you do this.
If the top view doesn't seem to have the correct symmetry, or the side view seems obviously wrong, then this technique may not work in your case. This problem can occur for one of several reasons. First, your particle may have a preferred orientation, and there may simply not be any top views present in the data set. Second, the image contrast might simply be too low for an accurate symmetry search (in this case, you might consider trying a higher dose, or negative stain for an initial model). Finally, the particles may not be well-centered in the box. The symmetry search can tolerate some degree of poor centering, but too much can make it fail.
If this method fails, you can try specifying 'unk' for the symmetry and following the resulting instructions, though for the subsequent steps (running the 'refine' command), you should specify the correct symmetry again). $dsym
startcsym start.hed #-ptcl-to-use sym=cn
Replace n with the symmetry of your particle. Note that you specify C symmetry for this program, even if your particle has D symmetry. '#-ptcl-to-use' is the number of particles used to generate the top and side views. This number should generally be between 20 and 100, and should not be more than 5-10% of your total number of particles. A larger number will give less noisy class-averages, but they will be blurred out by averaging a wider range of different orientations. A smaller number will give a sharper but noisier result. If you have a LOT of particles (>5000), you might want to use only the first few thousand for this step to save time, eg:
proc2d start.hed tmp.hed last=3999 startcsym tmp.hed 60 sym=c4 rm tmp.hed tmp.img
After running this command, several new files will exist:
cls000.hed - the individual particles used to generate the top view
cls001.hed - the individual particles used to generate the side view
classes.hed - contains 2 class averages, the top view and the side view
sym.hed - contains 3 images, the symmetrized top view, the side view, and
the side view aligned to the symmetric axis
threed.0a.mrc - The preliminary 3d model.
You might want to take a look at classes.hed and sym.hed. Make sure that the symmetrized and unsymmetrized top views look somewhat similar. Make sure the side view isn't too ridiculous. If these are ok, chances are, the 3d model is ok to try refining. Don't bother looking at the 3D model in projection. It will look terrible from most angles. An isosurface rendering will give you a better idea.
There is a possibility that the orientation of the side view may be determined incorrectly (the 3rd image in sym.img). Usually, if startcsym is wrong, it will be off by exactly 90 degrees. If this occurs, run:
startcsym start.hed #-ptcl-to-use sym=cn nosym fixrot=90
This will fix the 3D model withour rerunning the symmetry search. Double-check that it worked properly if you do this.
If the top view doesn't seem to have the correct symmetry, or the side view seems obviously wrong, then this technique may not work in your case. This problem can occur for one of several reasons. First, your particle may have a preferred orientation, and there may simply not be any top views present in the data set. Second, the image contrast might simply be too low for an accurate symmetry search (in this case, you might consider trying a higher dose, or negative stain for an initial model). Finally, the particles may not be well-centered in the box. The symmetry search can tolerate some degree of poor centering, but too much can make it fail.
If this method fails, you can try specifying 'unk' for the symmetry and following the resulting instructions, though for the subsequent steps (running the 'refine' command), you should specify the correct symmetry again).. $icos
Please note that, while EMAN does support icosahedral symmetry, the current version is better suited for lower symmetries. For particles with such high symmetries, other techniques may work better than the one used by EMAN. We have successfully used EMAN to refine several icoshedral particles, so it's useable, just remember that it's one of the more poorly supported features in the current version. We are actively working on optimizing these routines for future releases. You might consider using SAVR, an EMAN/MRC hybrid available on the NCMI home page.
There are many ways to generate a preliminary model from icosahedral data. EMAN uses a particularly simple and fast routine that will work well in many cases. The program starticos searches the data set (start.hed) for particles with the best 5, 3 and 2-fold symmetries. It will locate a number of particles for each symmetry, then align/average them to make a class average for each symmetric axis. These 3 views are then used to generate a 3d model. This routine will generally work well as long as the distribution of orientations contains particles near all 3 symmetries. If one or more is under-represented in the data, the result may not be satisfactory. Use it like this:
starticos start.hed #-ptcl-to-use [imask=radius]
The #-ptcl-to-use should generally be between 10 and 100, and less than 5-10% of the total number of particles. imask is optional. If your particle has a lot of non-icosahedral mass in the center (like DNA/RNA), this option allows you to exclude the center of each particle from the symmetry search. This may produce better results in some cases. Specify the exclusion radius in pixels if desired.
After running this command, several new files will exist:
cls000.hed - the individual particles used to generate the 5f view
cls001.hed - the individual particles used to generate the 3f view
cls002.hed - the individual particles used to generate the 2f view
classes.hed - contains 3 class averages, the 5f, 3f and 2f views
sym.hed - Contains the symmterized class averages
threed.0a.mrc - The preliminary 3d model.
You might want to take a look at classes.hed and sym.hed. Make sure that the symmetrized and unsymmetrized views look somewhat similar. If these are ok, chances are, the 3d model is ok to try a refinement on. $asym
I'm sorry to see that you're reading this section. Asymmetric particles can be among the most difficult particles to refine. Of course, this is not necessarily the case, certain particles, like ribosomes, actually refine very nicely using standard techniques with relatively little effort. However, other particles, like alpha-crystallin, are virtually impossible to refine to a single self-consistent answer. In general, if the particle has considerable internal structure OR has a very clear non-spherical shape, you're probably ok. In general if your particle has no symmetry at all, you must be very cautious when you reach the post-processing phase. You may need to perform some additional tests, such as radial randomization of the model with re-refinement. If there is any possibility that your particle has a pseudosymmetry, or may be vaguely cylindrical in shape, you may consider generating a starting model assuming some C symmetry.
Assuming you're going to stick it out and do a fully asymmetric starting model, you will first, use the results from the 'refine2d.py' command you ran in step 1.
Manually examine the class averages in iter.final.hed. Select some class averages that look good, and put them in a file called good.hed. You can do this by running 'v2 iter.final.hed', middle-clicking on the resulting view, then using 'split' mode to make a new view with selected particles in it. Then middle-click on the new view and save the particles into a new file. The precise number of classes to select depends on the particle. Generally at least 7 or 8 should be selected. You can take as many more as you like, but try to avoid particularly noisy ones, or duplicates of the same view. Once you have good.hed prepared, run:
startAny good.hed [sym=symetry]
If your particle has some symmetry, but was not amenable to the other methods, then specify it above. For asymmetric particles, don't specify sym=. $unk
If you don't know the symmetry of your molecule, it's best to try to make a preliminary guess, then relax the symmetry later if it proves to be incorrect. First, use the results from the 'refine2d.py' command you ran in step 1.
Manually examine the class averages in iter.final.hed. Select some class averages that look good, and put them in a file called good.hed. The precise number of classes to select depends on the particle. Generally at least 7 or 8 should be selected. You can take as many more as you like, but try to avoid particularly noisy ones, or duplicates of the same view. You can generate a new file with only the 'good' images in them as follows:
Simply close the 'Big View' when you're done with it. You might want to examine good.hed in the browser, just to make sure it contains all the images you wanted in the correct order.
The next step is to take these images and try to build a 3D model from them. This is accomplished with the 'startAny' command, as follows:
startAny good.hed [sym=c<n>] [proc=<nproc>]
Specify symmetry if it is known, but only a single c symmtery is allowed in this program. This will run for some time. It uses cross-common lines in Fourier space to try to determine the relative orientations of the particles, then eventually builds a 3d model. As with the other startup programs, the output will be written to threed.0a.mrc. When it's done, you might want to have a look at this model in projection or as an isosurface, to see if it came out well. To look at it in projection, either select it in the eman file browser, and rotate it with the right mouse button, or use v4 threed.0a.mrc. For isosurfaces, use your favorite MRC capable rendering program. Vis5d is included with the EMAN package. It's an excellent (and free) package originally designed for weather visualization. To use it, type:
mrc2v5dt threed.0a.mrc x.v5d vis5d -box 1 1 1 -path / x.v5d
Read the vis5d reference manual referenced above for more info. $2
You should now have start.hed/img and threed.0a.mrc. This is what you need to start a refinement. Go on to step 3.