Hello,
I am processing a helical filament formed by bacterial tubulin-like proteins. The expected structure consists of four distinct protofilaments arranged helically along the filament axis.
I previously processed this dataset using CryoSPARC v4.0.1. The reconstruction obtained in v4.0.1 showed the expected architecture, with four clearly separated protofilaments following a helical path.
After upgrading to CryoSPARC v5.0.6, I reprocessed the same dataset several times. However, the reconstruction consistently converged to a tube-like or cylindrical density. Instead of four separated protofilaments, the densities of the four protofilaments appear to merge laterally into a continuous cylinder.
The same raw dataset was used in both versions, and I attempted to keep the processing workflow and major parameters as similar as possible. The main difference is the CryoSPARC version.
I would like to ask:
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Were there any changes to the helical reconstruction or refinement algorithms between v4.0.1 and v5.0.6 that could affect this type of reconstruction?
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Are there any new default parameters or settings in v5.0.6 that should be carefully checked when processing an unusual helical assembly with only four protofilaments?
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Could the cylindrical reconstruction result from changes in helical symmetry optimization, regularization, initial model handling, or alignment settings?
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Has anyone observed a similar difference between CryoSPARC v4 and v5 when processing helical filaments?
The approximate helical parameters of this specimen are:
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Helical rise: approximately 79–82 Å
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Helical twist: approximately −5.5°
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Number of protofilaments: four
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Protein type: bacterial tubulin-like protein
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Expected morphology: four separated protofilaments forming a helical tube
Thank you.