asymmetric reconstruction
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I found a general general description for doing a virus asymmetric reconstruction with frealign here:http://grigoriefflab.janelia.org/node/2014.
Does anyone have any information on how to use the asymmetry scripts mentioned on that link to do a phage reconstruction? I've already completed the symmetric reconstruction with Frealign and I have an initial model from Auto3DEM.
I general description can be
I general description can be found in "Frealign: An exploratory tool for single-particle cryo-EM."
All of my scripts currently
In reply to I general description can be by niko
All of my scripts currently utilize frealign version 8.09 format.
Will Frealign 9.11 continue an iteration from version 8.09?
Do the methods mentioned in the ms (lowercase symmetry letter and mask) work with version 8.09?
The asymmetric refinement is
In reply to All of my scripts currently by rmcnulty
The asymmetric refinement is only available in v9.11. You should be able to switch to v9.11 with input files generated by v8.09.
I converted the v8.09
In reply to The asymmetric refinement is by niko
I converted the v8.09 parameter file to v9.11. The icosahedral reconstruction has slightly improved.
I have an icosahedral virus with a tail only at one of the 12 five folds. How can I get Frealign to align and reconstruct the asymmetric feature only at one five-fold? So far, I've tried "i1" but the tail doesn't show up. Next I planned to use my model containing a tail with a mask around the tail density. But I'm not sure this will orient the particles with the tail aligned to one 5-fold.
You need to create a 3D mask
In reply to I converted the v8.09 by rmcnulty
You need to create a 3D mask file that includes only one of the five subunits on one of the 12 vertices. Use this mask (key word mask_file in mparameters) with i1 symmetry to do a classification in which parameter alignment is turned off. To turn off parameter alignment, set all the flags in the parameter_mask (in mparameters) to zero, i.e. the string should be "0 0 0 0 0" instead of "1 1 1 1 1".
There are 7 proteins in the
In reply to You need to create a 3D mask by niko
There are 7 proteins in the icosahedral asymmetric unit. I made a mask around the protein that makes up one fifth of the five-fold, from only one of 12 vertices. I requested i1 and set parameter mask to 0 0 0 0 0. This is the error I get before the crash:
PGFIO-F-217/list-directed read/unit=5/attempt to read past end of file.
File name = stdin formatted, sequential access record = 11
In source file apply_mask.f, at line number 191
apply_mask.com finished
You changed the wrong "mask"
In reply to There are 7 proteins in the by rmcnulty
You changed the wrong "mask" parameters. Please change the parameters on the line that starts with the word "parameter_mask".
I've tried it with and
In reply to You changed the wrong "mask" by niko
I've tried it with and without parentheses. Always the same error.
parameter_mask 0 0 0 0 0
Can I please email you my mparameters_run file?
You have to use "" in this
In reply to I've tried it with and by rmcnulty
You have to use "" in this case.
Reggie, you should make sure
In reply to You have to use "" in this by niko
Reggie, you should make sure that your syntax is correct for the mparameters file (compare with the template mparameters) and that your mask is appropriately centered in the area of interest. You can come by Salk at some point if it doesn't work, and someone in the group can show you. This approach should work well for what you want to do.
I found the issue by running
In reply to You have to use "" in this by niko
I found the issue by running apply_mask.exe directly. The program ask for a couple of other items not in my mparameters file – like mask_edge. Once these items were added (also mentioned in your paper) the refinement was able to proceed.
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