R2 1 holey carbon film grids

For cryo-EM grid preparation, 1.5 µL of fibril solution were applied to freshly glow-discharged R2/1 holey carbon film grids (Quantifoil). After the grids were blotted for 12 s at a blot force of 10, the grids were flash frozen in liquid ethane using a Mark IV Vitrobot (Thermo Fisher) operated at 4 °C and 95% rH.
Cryo-EM datasets were collected on a Titan Krios transmission-electron microscope (Thermo Fisher) operated at 300 keV accelerating voltage and a nominal magnification of 81,000× using a K3 direct electron detector (Gatan) in non-superresolution counting mode, corresponding to a calibrated pixel size of 1.05 Å. Data was collected in EFTEM mode using a Quantum LS energy filter at a slit width of 20 eV. A total of 11,740, 7836, and 7744 movies were collected with SerialEM^{43 (link)} for Datasets 01, 02 and 03, respectively. Movies of Dataset 01 were recorded over 50 frames accumulating a total dose of ~51 e⁻/A², whereas movies of Dataset 02 and 03 contained 40 frames with a total dose of ~43 e⁻/A². The range of defocus values collected spans from −0.5 to −2.0 μm. Collected movies were motion corrected and dose weighted on-the-fly using Warp⁴⁴ .

Synchronized T. pseudonana cells were vitrified by plunge-freezing on glow discharged 200 mesh copper R2/1 holey carbon film grids (Quantifoil Micro Tools GmbH, Grossloebichau, Germany). In a Leica EM GP (Leica Microsystems GmbH, Wetzlar, Germany), 1 µl of artificial seawater was pipetted on the copper side in order to enhance media flow to the blotting paper and 4 µl of cell suspension at 7–13 × 10⁶ cells/ml was pipetted on the carbon side. The grids were blotted for 6 seconds from the back side of the grid before they were plunged into a liquid ethane bath cooled by liquid nitrogen.

For cryo-EM grid preparation, 1.5 µL of fibril solution was applied to freshly glow-discharged R2/1 holey carbon film grids (Quantifoil). After the grids were blotted for 12 s at a blot force of 10, the grids were flash-frozen in liquid ethane using a Mark IV Vitrobot (Thermo Fisher).
Cryo-EM data sets were collected on a Titan Krios transmission electron microscope (Thermo Fisher) operated at 300 keV accelerating voltage and a nominal magnification of ×81,000 using a K3 direct electron detector (Gatan) in non-super-resolution counting mode, corresponding to a calibrated pixel size of 1.05 Å. Data acquisition was done in EFTEM mode using a Quantum LS (Gatan) energy filter set to a slit width of 20 eV. A total of 14,417 movies were collected with SerialEM^{48 (link)}. Movies were recorded over 40 frames accumulating a total dose of ~40.5 e⁻/A². The range of defocus values collected spans from −0.7 to −2.0 μm. Collected movies were motion-corrected and dose-weighted on the fly using Warp^{49 (link)}.