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Translational Alignment

Since the HIV VLPs are not true spheres, our initial positions are quite rough. This is particularly true for the radial position (Z-axis in the subtomograms). In this step, we will perform a quick translational alignment with no angular search; this will improve the radial density in our reference, which will allow us to generate a tighter reference mask.

ccmask Creation

A cross-correlation mask (ccmask) is used to restrict the particle shifts during alignment. For this dataset, there is potentially a large error in the Z-direction, but error in the XY-plane is well defined. The appropriate shape of mask for this type of error is a cylinder. Since we seeded our positions at half the inter-subunit spacing, this is the maximum XY error and will be the radius of the mask.

Run this command in the STOPGAP Console to save a cylindrical mask 4 pixels wide and 24 high:

ccmask = sg_cylinder(32, 4, 24);
sg_mrcwrite('masks/ccmask.mrc', ccmask);

NOTE: A ccmask should always be binary! Do not use any Gaussian dropoff.

Run Translational Alignment

  1. Open the subtomo parser. Update the subtomo_mode to 'ali_singleref'.

  2. Set the angular search parameters. STOPGAP has multiple search strategies, with overlapping parameter sets. For now, set search_mode to 'hc', search_type to 'cone', and cone_search_type to 'coarse'. Since we don’t want to do any angular search for this iteration, set angincr, angiter, phi_angincr, and phi_angiter to 0.

  3. Set the bandpass filter settings. In general, the high pass filter defaults (hp_rad=1, hp_sigma=2) is fine; this mainly suppresses any normalization issues with the central voxel in Fourier space. An lp_sigma of 3 is usually fine. More important is to keep track of the low-pass filter radius (lp_rad) during your run. A rule of thumb is to make sure the lp_rad is less-than or equal to the Fourier radius where FSC=0.5. Since we don’t really have any resolution in our map, we can arbitrarily set it to 60 Å for now. STOPGAP sets filter values in Fourier pixels since real-space values do not round well, particularly for small boxsizes or high binnings. You can covert resolution to Fourier pixels with:

    In our current settings we have a 32 pixel boxsize and a 10.8 Å pixelsize so 60 Å resolution corresponds to 5.76 Fourier pixels. Since we cannot set fractional pixels, set lp_rad to 6, which corresponds to a resolution of 57.6 Å.

  4. Run the parser and run STOPGAP.

  5. Check ref_2.mrc in 3dmod. After this alignment, we now have the 3 layers we saw in the tomograms. (Use the XYZ or isosurface view.) Despite no angular alignment, we also already have some resolution of the in-plane structure.