Wrong output after local motion correction



Dear all,
HI, I recently collected some data on Talos Arctica, the pixel size in super resolution mode is 0.4 angstrom. First I used relion v3.0 for particle picking & classification using binned 2 data set (pixel size 1.6 angstrom). Then I import the final selected particles.star into cryosparc v2.5, and managed to get a 4.1 A resolution structure. To further improve resolution. I tried the local motion in cryosparc, here’s what I’ve done:

  1. re-extract all particles in relion to create a unbinned (pixel size 0.8) particles.star, then change the suffix for all micrograph name in particles.star to ‘_rigid_aligned.mrc’ to correspond to cryosparc name convention.
  2. import the raw movie into cryosparc, do motion correction
    3.import the changed particle,star into cryosparc, on the micrograph tab of this job, drag the previously completed motion correction output into the tab, then check the ignore raw data tab.
  3. use the imported particle location and the cryosparc motion correction result to do local motion job.
  4. since the cryosparc motion correction output has a pixel size of 0.4 angstrom, downsample the local motion output by a factor of 4, resulting a pixel size of 1.6 angstrom.
  5. use the downsample job output to do refinement.
    And the output was totally wrong, didn’t have a right shape of the molecule.
    Any idea what went wrong during the whole process?
    Thank you very much in advance!