Ceta camera

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.

For negative stain TEM observations (Fig. 5n–q), 3 μL of sample were applied to the clean side of carbon on a carbon-mica interface and stained with 2% sodium silicotungstate. Micrographs were recorded on a FEI Tecnai T12 microscope operated at 120 kV with a Gatan Orius 1000 camera, at a nominal magnification of ×49,000 resulting in a pixel size of 1.26 Å. For cryo-EM observations, 3 µL of sample were applied to glow-discharged quantifoil grids 300 mesh 1.2/1.3 (Quantifoil Micro Tools GmbH, Germany), excess solution was blotted with a Vitrobot (FEI) and the grid frozen in liquid ethane^{58 (link)}. Data collection was performed on a FEI F20 microscope operated at 300 kV under low dose conditions. Images were recorded on a CETA camera (FEI) at a nominal magnification of ×50,000 corresponding to a pixel size of 2.09 Å. Images were converted into the TIFF format and then imported into the software ImageJ v1.51k^{41 (link)} to calculate the length and curvature of oligomers.

A 4-μl drop of freshly assembled RNP-like diluted to 0.05 mg/ml was applied to the clean carbon side of a carbon/mica interface. The sample on carbon was then immediately stained using a 2% (w/v) sodium silicotungstate (pH 7.0), 200-μl drop, fished with a 400-mesh copper grid (Quantifoil), and air-dried at room temperature. Images were collected on a Tecnai F20 electron microscope (FEI Tecnai, Hillsboro, OR, USA) operating at 200 kV at a nominal magnification of ×55,000 and equipped with an FEI Ceta camera.