Reconstruction of asymmetric components in icosahedral structures

A practical approach is to first process the data by ignoring the presence of any asymmetric component, thus just exploiting the symmetric properties of the structure. Since a symmetrized reconstruction will be used at each iteration, determination of the orientation and centre for each particle is expected to be sufficiently accurate, under the assumption that the additional component only minimally affects such determination. Once the icosahedral reconstruction does not improve after further iterations, you can follow the following procedure:

  • Generate a suitable template
    Generate a reference map containing additional density, placed at just one equivalent site where the component is expected. The density can be generated synthetically, or by merging the icosahedral reconstruction with the density from some available reconstruction of the component. Generally it does not need to be a faithful representation of such component.
  • Modify the parameter file
    Modify the last parameter file generated by Auto3DEM (file ending with _continue), possibly saving it with a different name for bookkeeping purposes.
    • change the reference map by pointing the following parameters to the asymmetric template
      auto start_map
      auto start_map_even
      auto start_map_odd
    • release the constraint of icosahedral symmetry by deleting the line
      auto symm_code 532
    • don't apply any radial mask
      auto hollow_map 0
      Alternatively you can set an optimal radial range by keeping the parameter set to 1, and setting manually the values of
      auto hollow_in_rad
      auto hollow_out_rad
      to include all the asymmetric reconstruction, after making sure that
      auto hollow_auto 0
    • avoid solvent flattening of the map to assure that the asymmetric feature is fully included in the reference
      auto flatten_map 0
    • set the refinement to search only among the 60 solutions equivalent to the one previously determined
      po2r mode ticos_equiv
    • fix the center coordinates of the particles to reduce the risk of bias
      po2r ncenter 0
    • use only low resolution features to determine the best orientation
      po2r res_max 40
      The value of 40 Å is just indicative, although a good start, and can be changed to reflect the size of the asymmetric component.
    • keep the resolution range constant from one iteration to the next
      auto freeze_res 1
    • generate a reconstruction, after the refinement, without imposing any symmetry
      p3dr symm_code 1
  • Generate an initial asymmetric map
    Using the modified parameter file, run 3 to 5 iterations, until the reconstruction does not change.
  • Refine the reconstruction
    Run additional iterations after modifying the last parameter file generated by Auto3DEM by changing back few parameters
    auto freeze_res 0
    po2r mode local
    po2r symm_code 1
    po2r ncenter 4
    auto flatten_map 1 (optional)
    Additionally, set
    auto res_max
    to a resolution value close but larger than the resolution estimated by the program for the latest asymmetric reconstruction.