Inverted data sign after 3D flex of local motion corrected particles

Hi @team,

just realized an unexpected behavior and wanted to confirm my procedure.
Obviously, particles still have flipped data sign after local motion correction, which was never an issue during downstream refinement.
When I tried 3dflex, training ran smoothly (as far as I can tell), flex generate provided comprehensible series. Due to memory issues (400k particle images, 448 px box) I needed to downsample particles to 384 px. Flex reconstruct obviously failed on these.

After flipping sign of the downsampled particles (now white on dark) reconstruct succeeded, creating inverted reconstructions however.

Is data sign indeed manipulated during flex prep? Couldn’t find anything in the docs.
Anything else to consider for the input particles?


Hi @tarek!

Thank you for bringing this topic up. Particles after Local Motion Correction retain the same sign as the upstream processing, and your intuition is correct: the Flex Data Prep job performs a signal flip where necessary, depending on the sign of the input particle images. If particle images from a Downsample job (without subsequent Flex Data Prep) are used for Flex Reconstruct then they may result in an inverted volume, as you observed. We would generally recommend that any particles used for Flex analyses (Training or Reconstruction) come from a Flex Data Prep job.

Hopefully that resolves your questions about downsampling for Flex Reconstruction!