Talos arctica microscope

Vitrified specimens were prepared by adding 4 μl samples of rpf2Δ255-344 or GAL-SDA1 particles at a concentration of ~150 nM to a glow-discharged holey carbon grid (Quantifoil R2/2) covered with a freshly made thin carbon film. Grids were blotted for 1 s and plunge-frozen into liquid ethane using an FEI Vitrobot Mark IV (4 °C and 100% humidity). The cryo-grids were initially screened at a nominal magnification of ×92,000 in an FEI Talos Arctica microscope, operated with an acceleration voltage of 200 kV. Good-quality grids were transferred to an FEI Titan Krios electron microscope that was operating at 300 kV and images were recorded using a K2 Summit direct electron detector (Gatan) in counting mode at a nominal magnification of ×130,000, corresponding to a pixel size of 1.057 Å at the object scale and with the defocus varying from −1.0 to −2.0 μm. Coma-free alignment was manually optimized and parallel illumination was verified before data collection. All micrographs obtained with the K2 camera were collected semi-automatically by SerialEM^{53 (link)}, under low-dose conditions. Each micrograph was dose fractionated to 32 frames with a dose rate of ~8.0 counts per physical pixel per second for a total exposure time of 8 s.

Holey-carbon grids (Quantifoil, R1.2/1.3 with 2 nm C) were coated with pentylamine (Sigma-Aldrich, 171409) using a method described earlier^{9 (link)} and glow-discharged with a plasma cleaner. In total, 3 µl of the freshly prepared sample at a final concentration of 300–500 nM was applied to the grid and blotted for 1.5 s before vitrification in liquid ethane precooled by liquid nitrogen. Cryo-grid preparation was assisted by an automated plunge freezer (Leica Microsystems) with the inner chamber set at 15 °C and 90% humidity. The cryo-grids were prescreened with a 200 kV FEI Talos Arctica microscope (FEI Falcon II camera). Final high-resolution datasets were collected on a 300 kV FEI Titan Krios TEM (Gatan K3 summit camera) with GIF Quantum energy filter (Gatan). The images were collected at a dose rate of 0.97e⁻ s⁻¹ Å⁻² and with an exposure time of 3 s. Movie stacks (50 frames each) were recorded under super-resolution conditions. The magnification was set at ×105,000, and the defocus ranged from −0.7 μm to −2.2 μm. Statistics for data collection are summarized in Extended Data Table 1.

Holy-carbon gold grids (Quantifoil, R1.2/1.3) were treated with Solarus 950 plasma cleaner (Gatan) with a 4:1 ratio of O₂/H₂ for 60 s for glow-discharge before cryo-EM sample preparation. 4 μL aliquots of freshly prepared PAPP-A·proMBP or PAPP-A·STC2 (0.2 mg/mL) complex were applied on the grids, blotted with filter paper (Whatman No. 1) with force set to –2 for 0.5 s at 4 °C and 100% humidity, and plunge-frozen in the liquid ethane using a Vitrobot Mark IV (FEI).
The cryo-grids were screened on a 200 kV Talos Arctica microscope equipped with an FEI Ceta camera and a K2 Summit direct electron detector (Gatan). Data collection was carried out with Titan Krios electron microscope (FEI) operated at 300 kV.
Images were recorded with a K2 Summit direct electron detector (Gatan) in the super-resolution mode at a nominal magnification of 130,000× and a dose rate of 8 e⁻/s/pixel. Movies were recorded semi-automatically using the SerialEM software^{62 (link)}. A GIF Quantum energy filter (Gatan), with a slit width of 20 eV was used at the end of the detector. The defocus range was set from –0.7 to –1.2 μm. The total exposure time was 8.32 s, and intermediate frames were recorded every 0.26 s. 32 frames per image were acquired. Statistics for data collection are summarized in Supplementary Table S1.