GoKawiilSample preparation for cryo-ET
Cryo-ET samples were prepared using 200-mesh R2/2 copper grids (Quantifoil) plasma-cleaned for 30 s in 75% argon/25% oxygen using a 1070 plasma cleaner (Fischione). S. pombe cells were grown for 7 days in EMM containing 0.5% (w/v) glucose and diluted in glucose-free EMM to OD 600 = 0.6 immediately before freezing. A 4 µl aliquot was applied to the carbon side of the grid and back-side blotted for 1 s (Whatman 597 paper; paper contact sensing mode with 1.5 mm movement) using a Leica EM GP2 plunger operated at 23 °C and 100% humidity. Grids were vitrified in liquid ethane.
Cryo-FIB milling
Vitrified grids were mounted in Cryo-FIB Autogrids (ThermoScientific, 1205101) under liquid nitrogen and transferred to an Aquilos 2 cryo-FIB/SEM (ThermoScientific). The stage and anti-contamination shield were maintained at −194 °C. After scanning electron microscope screening (5 kV, 13 pA), grids were coated with trimethyl(methylcyclopentadienyl)platinum(IV) (GIS, 120 s), preceded and followed by 15 s sputter coating (30 mA). Positions were set using MAPS, and lamellae were milled using AutoTEM (30 kV; 1 nA to 30 pA) and thinned manually to ~160 nm at 30 pA. During final thinning, the stage was tilted by +0.5° to improve surface polishing and reduce curtaining. A final sputter coating (3 s, 10 mA) reduced charging during transmission electron microscopy acquisition and facilitated tilt-series alignment.
Mitochondria-focused cryo-ET acquisition
Data collection
To initially assess ribosomes proximal to mitochondria, lamellae were screened and tilt series were targeted to mitochondrial regions. Data were collected on a Titan Krios G4i (ThermoScientific) operated at 300 kV, equipped with an energy filter and Falcon4i detector in zero-loss mode. Lamellae were mapped at 14.39 Å per pixel (−70 µm defocus; 70 µm objective aperture; 20 eV slit). Mitochondria were selected in SerialEM44, guided by SPACEtomo segmentation and PACEtomo45 for beam image-shift parallel acquisition. Tilt series were acquired at 64,000× (1.933 Å per pixel), using a 50 µm C2 and 70 µm objective aperture (10 eV slit), in nanoprobe mode (spot size 5; 1.4 µm illumination). A dose-symmetric scheme (3° increments, −65° to +49°, centred on ~−8° pre-tilt) was used. Movies were recorded in counting mode (880 ms; ~3.34 e− Å−2 per tilt; total dose ~130 e− Å−2). Target defocus ranged from −2 to −6 µm. Seventy-eight tilt series were selected for subtomogram averaging based on dose, thickness, contamination and cellular integrity.
Data processing
Stage tilts in .mdoc files were corrected for lamella pre-tilt. EER frames were converted to TIFF (RELION v4.0.146), motion-corrected, and contrast transfer function (CTF)-estimated (−2 to −10 µm) in Warp 1.0947. After manual inspection, tilt series were aligned in AreTomo248, defocus handedness verified, and 3D-CTF–corrected tomograms reconstructed at 15.44 Å per pixel in Warp.
Ribosome picking and classification
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