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EMAN Reconstruction - Phase 3
In this step we seek to refine our preliminary model to generate a final,
(hopefully) high resolution, structure.
1. Run the 'refine' command.
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At this point you only need 2 files your working directory. If you'd like
you can remove the others, but it's not really necessary. start.hed/img
contains the raw particle data (preprocessed with ctfit), and threed.0a.mrc
contains the preliminary 3d model you generated in the last step. The
refine
command is fairly complecated and has a lot of options. For now, we'll start
with a basic refine command which should work well in most cases. Generally it's
a good idea to run this command from a
screen window, or submit
it as a background job with nohup and '&' (see man page). You can monitor the
progress of the run with eman, by looking at the command history.
refine will run numerous other eman programs, much like a script, while
it executes. Each of these individual programs will appear in the program history.
The end of one refinement iteration is marked by the completion of a proc3d
command.
Here's the refine command to run for your particle:
$1n
At this point you only need 2 files your working directory. If you'd like
you can remove the others, but it's not really necessary. start.hed/img
contains the raw particle data (filtered earlier), and threed.0a.mrc
contains the preliminary 3d model you generated in the last step. The
refine
command is fairly complecated and has a lot of options. For now, we'll start
with a basic refine command which should work well in most cases. Generally it's
a good idea to run this command from a
screen window, or submit
it as a background job with nohup and '&' (see man page). You can monitor the
progress of the run with eman, by looking at the command history.
refine will run numerous other eman programs, much like a script, while
it executes. Each of these individual programs will appear in the program history.
The end of one refinement iteration is marked by the completion of a proc3d
command.
Here's the refine command to run for your particle:
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If you have a good feel for the size of your particle, you can replace the '-1'
in mask=-1 with a safe mask radius for your macromolecule.
Look at the refine documentation for info
on all of the parameters, but there are a few basics you should be aware of:
- The 'sep=2' option will cause each particle to be included in the 2 best
matching classes. This generally helps handle the problem of uncertainty in particle
classification. Since nearly 1/2 of the data will be thrown out during alignment/averaging,
this shouldn't pose any real problem. If you disapprove of this, remove 'sep=2' and
increase classkeep to ~1.
- The 'tree=2' or 'tree=3' options (which may or may not be in the command above) are
used to help classification run faster. See the refine man page for details.
- The 'euler2=' option is not included by default. You might experiment with using
'euler2=1' or 'euler2=2', and examining how the Euler angle distribution changes in
your 'classes.#.img' files. This option reassigns euler angles to the class averages
using projection matching. If you specify euler2=2, projections will be generated
with ang/2 angular spacing (roughly 4x as many projections).
- The '3dit' and '3dit2' options are used to eliminate artifacts produced by
using direct Fourier methods for building 3D models. Both options iteratively try
to make the models better match the class averages in real space. The '3dit2' option
also somewhat aggressively tries to zero any mass that isn't strongly defined in
the data. If you use too many iterations of '3dit2', you will end up with a model
which is very consistent to very high resolution, but any questionable features
(including some real ones) will be eliminated. This only occurs for large 3dit2 values,
(larger than ~4).
- The single number following refine tells the command how many refinement
iterations to run. For each iteration, a classes.n.hed/img, a
threed.n.mrc, a threed.na.mrc, and sometimes an x.n.mrc
file will be generated (as well as a bunch of temporary files which are
overwritten each iteration). If you kill the refine job while it's running, you
can restart it roughly where it left off just by running the same command again.
The number of iterations is the TOTAL number of iterations to run, not the
number of additional iterations. That is, if your run refine 5 ... for 3
iterations, then kill it and run refine 5 ... again, it will only do
2 more iterations. If you run a couple of iterations, and decide to change
some of the parameters, it's ok to kill the job, then run it again with the
new parameters. At most, you will loose 1/2 an iteration of processing time. If
you want to restart the refinement from scratch, you MUST delete all of the
classes.*.hed/img files AND all of the threed.*.mrc files (EXCEPT
threed.0a.mrc).
There are also a number of new refine options in this version of EMAN. These options
are still considered experimental, but in some cases one or more may make a dramatic improvement
in refinement quality.
- ctfcw= If you had a structure factor file when determining CTF parameters, you should
use the ctfcw option instead of the ctfc option. Rather than a resolution, you specify the
filespec containing the structure factor. This will perform a true Wiener filter on the data using
the accurate contrast estimate possible when the structure factor is known. This will produce
much better results than ctfc=.
- tree=<2 or 3> This option will speed up a refinement, but doesn't actually improve the results.
This option will perform classification in 2 steps. First it will determine the Euler angles roughly,
then it will 'fine-tune' them. This can produce up to a factor of 10 speed increase in classification.
One warning, however, not all particles are well-suited to this sort of speed-up. In some cases, it
will cause some incorrect classifications. '3' will increase the speed more than 2, but should only
be used for small angular increments (ang=).
- amask=,, This option can eliminate virtually all artifacts outside the 3D model,
although there can be some risk of chopping off any very low density features that might exist near the
periphery of the model. This option uses the 'automask2' option in proc3d to generate a custom mask
around the 3D model. This also enables projection specific masking of class-averages. This option can
dramatically improve the convergence and resolution of a model, and can prevent the build-up of noise
based iterative artifacts. Note that to use this option, you MUST also use the 'xfiles=' option.
- usefilt This is a very new option in refine. Normally, the raw particles are used for
classification and alignment. However, for optimal results, actually these tasks should be performed
on filtered images (Wiener filters are one good choice). This option will assume you have generated
a file called 'start.filt.img' which contains filtered versions of the images in 'start.hed'. You
may use any filter you wish, but 'proc2d start.hed start.filt.hed wiener= hp=3' seems to
work well. Note that this type of filter requires a structure factor file as discussed above.
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