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EMAN Reconstruction - Phase 3
You can click here to open this file in
netscape (if it's running on the local machine).
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).
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