Cryo-EM was carried out on samples prepared by depositing 3 µL solution of PFK assemblies (0.2 mM peptide concentration) on glow-discharged Quantifoil R 1.2/1.3 holey carbon grids (Quantifoil Micro Tools GmbH, Germany). Samples were manually blotted for three seconds at room temperature following vitrification by rapidly plunging into liquid ethane using a home-built plunging apparatus. The frozen samples were stored in liquid nitrogen until imaging. For imaging the samples were loaded under cryogenic conditions and imaged in low-dose mode on a Glacios microscope (Thermo-Fisher) operated at 200 kV. Automated data collection was done using EPU (Thermo-Fisher). Movies were collected at a calibrated pixel size of 0.89 Å by a Falcon4i direct electron detector fitted behind a Selectris X energy filter (Thermo-Fisher) set to ±5 eV around zero-loss peak. The detector was operated in a dose fractionated counting mode, at a dose rate of 8 ē/pixel/second, with a total electron dose of 30 ē/Å 2 . Data was collected at a defocus range of -0.5 to -1.5 μm. All processing was done using cryoSPARC v4.3.0. 51 Dose-fractionated movie stacks were patch-motion corrected, and their defocus values were estimated. The aligned sum images were used for further processing. Particles were picked using filament tracer followed by extraction of 500 pix long overlapping segments (with a shift of 2 times of the expected axial rise).