Glacios transmission electron microscope

Immediately before sample preparation, gold C-flat grids (C-flat CF-1.2/1.3-4Au-50; Protochips) were glow discharged in a Pelco easiGlow for 30 s with a current of 15 mA. Grid vitrification was performed on an EM GP (Leica) at 90% humidity and 4°C. Before sample application to the grid, 2 mM GTP (final concentration) was added to either FL ATL2(1–583) or ATL2(1–547) and allowed to incubate for 30 s. A volume of 3 µl of the sample was then applied to the grid, held for 5 s, blotted for 4 s, and plunged into liquid nitrogen–cooled ethane.
Tilt series were collected on a 200-kV Glacios transmission electron microscope (Thermo Fisher Scientific). Images were recorded using a K3 (Gatan; 18 frames per image) or a Falcon 4 (Thermo Fisher Scientific; 8 frames per image) direct detector operating in the electron counting mode. Tilt series were recorded using SerialEM (Mastronarde, 2005 (link)) software at a magnification corresponding to a pixel size of 2.37 Å as individual TIFF files per tilt. A grouped dose symmetric tilt scheme was implemented starting at 0°, followed by ±3–9, ±12–18, ±21–27, ±30–36, and ±39–48 with a tilt increment of 3° at −5 µm underfocus. The total electron dose was kept in the range of 80–90 e⁻/Ų.

Both PIC-nucleosome complexes (∼130 μl) were incubated on a floating ∼3.0 nm continuous carbon support for 7 min, after which the carbon film was attached to a holey carbon grid (Quantifoil R3.5/1, copper, mesh 200), washed with 4 μl of PIC-dialysis buffer and placed in a Vitrobot Mark IV (FEI/Thermo Fisher Scientific) under 100% humidity at 4°C. Under these conditions, samples were blotted with force 5 for 2 s and plunged frozen into liquid ethane. Optimal samples were identified using a Glacios transmission-electron microscope (Thermo Fisher Scientific) operated at 200 keV and equipped with a Falcon-III direct-electron detector (Thermo Fisher Scientific). Data was then collected using SerialEM 4.0⁵⁴ (link) on a Titan Krios G2 transmission-electron microscope (FEI/Thermo Fisher Scientific) operated at 300 keV, with 20 eV slit width of a QuantumLS energy filter (Gatan), and equipped with a K3 summit direct detector. Imaging was performed at a nominal magnification of 81,000x (corresponding to a pixel size of 1.05 Å/pixel), with 3 s exposure in counting mode and a total dose of 41.58 and 50.45 e^- per Ų, over 40 and 50 frames for PIC-Nuc^18W and PIC-Nuc^10W, respectively, at a defocus range from 0.5-1.5 μm. A total of 41517 and 36478 micrographs were collected for PIC-nucleosome^18W and PIC-nucleosome^10W, respectively.

To verify the presence and size of MVs, 50 μL of the purified MVs was used for cryo-EM imaging. Plunge freezing and imaging was conducted at the Electron Microscopy Facility of the Vienna BioCenter on a Glacios transmission electron microscope (Thermo Fisher Scientific).

For cryo-EM sample preparation, P-Rex1 was used at a final concentration of 3 μM and n-dodecyl-β-D-maltoside (DDM) was added to a final concentration of 0.08 mM. For samples with IP₄, a final concentration of 40 μM IP₄ was added. A sample of 4 μl was applied to a glow-discharged Quantifoil (1.2/1.3) 300-mesh grid which was then blotted with filter paper and plunge-frozen into liquid ethane cooled with liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher Scientific) set to 4 °C, 100% humidity, 4 second blot, and a force of 10. Micrographs were collected either using Leginon (Suloway et al., 2005 (link)) on a Glacios transmission electron microscope (Thermo Fisher Scientific) operating at 200 keV and a K2 Summit direct electron detector (Gatan, Inc.) in counting mode (0.98 Å/pixel) at a nominal magnification of 45,000x or using EPU (Thermo Fisher Scientific) on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operating at 300 keV and a K3 direct electron detector (Gatan, Inc.) in counting mode (1.054 Å/pixel) at a nominal magnification of 81,000x. On the Krios, datasets were collected on both untilted and 30° tilted grids (Table 1).