EER defect & gain reference problem

Hi all, I had a problem and question about EER format movies on cryoSPARC patch-motion correction.
Recently, I collected data from a Krios microscope equipped with a Falcon-4i detector and obtained a gain reference file (.gain format file) from the Institute.

Then, I imported my movies into cryoSPARC (v4.2.1) with gain reference (no upsampling and final frame = 40), and I didn’t observe the black lines that are commonly reported with EER format files. However, I noticed that the gain reference file contain a white column of dotted lines, which may indicate defect points or zero signal points ? (Fig 1)

Regardless, the gain-normalized movies look good. I’m planning to proceed with Patch-Motion → blob pick → extract → 2D class in general.

However, I have encountered some peculiar 2D classes and their corresponding locations that I’ve never seen in my other cases using .mrc or .tiff formats.
These seem to align with the white lines present in the gain reference (Fig.2)

Fortunately, the Relion (v4) MotionCorr does not exhibit these peculiar 2D classes. Are there any significant differences in how cryoSPARC and Relion process EER format data?

I usually prefer cryoSPARC processing on my system, but in this situation on EER, I have to use Relion
Has anyone else encountered this problem?

Thank you!


  1. I attempted to generate a gain reference from images (~ 250 movies) using ‘relion_estimate_gain’, but encountered the same problem.

  2. I converted the .EER file to TIFF format using ‘relion_convert_to_tiff’, with both the original and generated gain reference, but the problem persisted.

  3. Local motion correction also same problem.

  4. full-frame motion correction (not yet check)


  1. Have you experimented with flips and rotations of the gain reference?
  2. Would you be willing to share with the Structura team a set of 100 eer files for testing?

Thank you for your response!

Yes. I’ve explored as many combinations of flips and rotations as possible. If the wrong gain flips or rotations were chosen, the quality of the movies was significantly degraded (ex. additional appearance of black lines or the loss of particle features due to the highly intense signal from the white defect line)

Sure! each .eer movie is ~ 700 MB in size, so 100 EER files would total around 70 GB. What would be an efficient way to share these files with the Structura team?

Other group’s data collection from the same beamline Krios have reported similar issues, but it’s not certain that this issue occurs with all Falcon-4i detectors since I only have experience with one. It could be a problem specific to the beamline detector.
Also, I didn’t receive a ‘Defect file’ from the Institute. Is it possible the ‘Defect file’ could be generating directly from the Falcon-4i/EPU or .eer files? I’ve never used a defect file during movie import.

I am hopeful to identify what might be causing these issues.
Thank you.