Icosohedral virus map with mirror symmetry?

Submitted by mcjohnson on Fri, 2015-05-01 14:57

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Frealign

We've been refining an icosohedral virus, and everything seems like it has been running normally, but the final map seems to have mirror symmetry along all symmetry axes... What could be causing this? The parameters we've been using are:

Control parameter file to run Frealign
======================================

This file must me kept in the project working directory from which the refinement scripts are launched.

Note: Please make sure that project and scratch directories (if specified) are accessible by all sub-processes that are run on cluster nodes.

# Computer-specific setting
cluster_type         PBS	! Set to "sge", "lsf", "slurm", "pbs" or "condor" when running on an SGE, LSF, SLURM, PBS or CONDOR cluster, otherwise set to "none"
.
nprocessor_ref       40		! Number of CPUs to use during refinement.
nprocessor_rec       40		! Number of CPUs to use during reconstruction.
mem_per_cpu	  1000		! Memory available per CPU (in MB).

# Refinement-specific parameters
MODE                  1		! 1, 2, 3 or 4. Refinement mode, normally 1. Set to 2 for additional search.
start_process         24 		! First cycle to execute. Output files from previous cycle (n-1) required.
end_process           25 	! Last cycle to execute.
res_high_refinement   7.3 	! High-resolution limit for particle alignment.
res_high_class        10.0	! High-resolution limit to calculate class membership (OCC).
thresh_reconst        16	        ! Particles with scores below this value will not be included in the reconstruction.
nclasses              1		! Number of classes to use.

# Search-specific parameters
res_search           10.0	! High-resolution limit for orientational search.
thresh_refine        50.0	! Mode 4: Score threshold above which search will not be performed.
DANG                200.0	! Mode 3 and 4: Angular step for orientational search.
ITMAX               200		! Mode 2 and 4: Number of repetitions of grid search with random starting angles.
Bsearch            2000.0	! B-factor filtering (when > 0) applied during search.

# Dataset-specific parameters
data_input           run1		! Root name for parameter and map files.
raw_images           ../stacks/start
image_contrast        N		! N or P. Set to N if particles are dark on bright background, otherwise set to P.
outer_radius        600.0	! Outer radius of spehrical particle mask in Angstrom.
inner_radius         415.0	! Inner radius of spehrical particle mask in Angstrom.
mol_mass           31500.0	! Molecular mass in kDa of particle or helical segment.
Symmetry             I  	! Symmetry of particle.
pix_size              3.24   	! Pixel size of particle in Angstrom.
dstep                 3.24	! Pixel size of detector in micrometer.
Aberration            2.7	! Sherical aberration coefficient in millimeter.
Voltage             300.0	! Beam accelleration voltage in kilovolt.
Amp_contrast          0.07	! Amplitude contrast.

# Expert parameters (for expert users)
XSTD                  0.0	! Tighter masking of 3D map (XSTD > 0) or particles (XSTD

niko Fri, 2015-05-01 15:21

It is possible that your reconstruction consists of particles inserted using the correct and the wrong handedness. This can happen when starting from a low-resolution reference map where the true handedness of the particle has not yet apparent. If the refinement never develops a clear handedness of the reference, it is difficult to discriminate particles inserted correctly from particles inserted according to the wrong hand. One possible way to separate particles with right and wrong hand is to run a classification with two classes. Just continue your refinement but set nclasses to 2. Hopefully you will end up with two reconstructions that have opposite handedness but do not have mirror symmetry anymore.