Icosohedral virus map with mirror symmetry?
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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 0). PBC 5.0 ! Discriminate particles with different scores during reconstruction. Small values (5 - 10) discriminate more than large values (50 - 100). parameter_mask "1 1 1 1 1" ! Five flags of 0 or 1 (e.g. 1 1 1 1 1). Determines which parameters are refined. refineangleinc 4 ! When larger than 1: Alternate between refinement of OCC and OCC + angles. refineshiftinc 4 ! When larger than 1: Alternate between refinement of OCC and OCC + angles + shifts. res_reconstruction 6.5 ! High-resolution limit of reconstruction. Normally set to Nyquist limit. res_low_refinement 1500.0 ! Low-resolution limit for particle alignment. Set to particle dimention or larger. FMAG F ! T or F. Set to T to refine particle magnification. Not recommended in most cases. FDEF F ! T or F. Set to T to refine defocus per micrograph. Not recommended in most cases. FASTIG F ! T or F. Set to T to refine astigmatism. Not recommended in most cases. FPART F ! T or F. Set to T to refine defocus for each particle. Not recommended in most cases. FFILT T ! T or F. Set to T to apply optimal filter to reconstruction. Recommended in most cases. FMATCH F ! T or F. Set to T to output matching projections. Only needed for diagnostics. FBEAUT T ! T or F. Set to T to apply symmetry also in real space. Not needed in most cases. FBOOST F ! T or F. Set to T to allow potential overfitting during refinement. Not recommended in most cases. RBfactor 0.0 ! B-factor sharpening (when 0) applied during refinement. Not recommended in most cases. mp_cpus 1 ! Number of CPUs to use for each reconstruction job. restart_after_crash F ! T or F. Set to T to restart job if it crashes. delete_scratch T ! Delete intermediate files in scratch directory. qsub_string_ref "" ! String to add to cluster jobs submitted for refinement (only for SGE and PBS clusters). qsub_string_rec "" ! String to add to cluster jobs submitted for reconstruction (only for SGE and PBS clusters). first_particle last_particle frealign_bin_dir scratch_dir # Janelia-specific parameters night_queue F ! T or F. Set to T to use night queue. reschedule_if_qw F ! T or F. Set to T to reschedule jobs on the normal queue if some end up waiting on night queue.
It is possible that your
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.
This fixed the problem
In reply to It is possible that your by niko
This fixed the problem (and gained us a bit more resolution, too).
Thanks!