Epu data acquisition software

Grids preparation and data collection were carried out in S²C² (Menlo Park, CA). The WT MelB_St/Nb725_4/NabFab/anti-NabFab Nb complex was thawed out from –80°C storage. Aliquots of 3 μL of samples at 0.75 mg/mL or 1.5 mg/mL protein concentration were placed on 300-mesh Cu holey carbon grids (Quantifoil, R1.2/1.3) after glow-discharge (PELCO easiGlow) at 15 mA for 30 s and blotted on two sides for 3 s at 4°C and 100% humidity before vitrified in liquid ethane using a Vitrobot Mark IV (Thermo Fisher). The grids were subsequently transferred to a Titan Krios (G3i) electron microscope (Thermo Fisher) operating at 300 kV and equipped with K3 direct electron detector (Gatan) and BioQuantum energy filter. The best grids were found from the sample at 1.5 mg/mL concentration.
A total of 14,094 and 8715 movies were automatically collected using the EPU Data Acquisition Software (Thermo Fisher) for non-tilted and 30° tilted collections, respectively. Both datasets were collected at a 0.86 Å pixel size and a dose of 50 e⁻/Å⁻² across 40 frames (0.0535 or 0.07025 s per frame for the non-tilted and tilted data collection, respectively) at a dose rate of approximately 23.36 or 17.8 e⁻/Ų/s, respectively. A set of defocus values was applied ranging from −0.8, to 1.0, −1.2,–1.4, or –1.8 μm with an energy filter slit width of 20 eV and a 100 μm objective aperture.

Samples of PAPP-A·STC2 were diluted to a concentration of 0.6 mg/mL. C-flat holey carbon grids, CF-2.2-4C (Protochips), were glow-discharged on a Quorum GloQube Plus glow discharge system at 15 mA for 45 s. 3 µL of protein sample were added to the grids and vitrified at 4 °C and 100% humidity with a blotting time of 4 s on an EM GP2 automatic plunge freezer (Leica). Movies were collected on a Titan Krios G3i (EMBION, Danish National Cryo-EM Facility, Aarhus node) with X-FEG operated at 300 kV and equipped with a Gatan K3 camera and a Bioquantum energy filter using a 20 eV slit width. Movies were collected using aberration-free image shift (AFIS) data collection with the EPU data acquisition software (Thermo Fisher Scientific) at a pixel size of 0.507 Å/pixel (corresponding to a magnification of ×165,000). Two datasets were collected. Dataset I contained 10,073 movies with 59 dose fractions over a 0.8 s exposure and a total dose of ~58 e⁻ per Ų. Defocus range was 0.6–1.6 over six exposures in each hole. Dataset II contained 32,115 movies with 67 dose fractions over a 0.91 s exposure and a total dose of ~59 e⁻ per Ų. Defocus range was 0.6–1.8 over seven exposures in each hole). Further details are given in Supplementary Fig. 2 and Table 1.

Grids preparation and data collection were carried out in S²C² (Menlo Park, CA). The WT MelB_St/Nb725_4/NabFab/anti-NabFab Nb complex was thawed out from −80 °C storage. Aliquots of 3 μL of samples at 0.75 mg/ml or 1.5 mg/ml protein concentration were placed on 300-mesh Cu holey carbon grids (Quantifoil, R1.2/1.3) after glow-discharge (PELCO easiGlow^™) at 15 mA for 30 sec, and blotted on two sides for 3 sec at 4 °C and 100% humidity before vitrified in liquid ethane using a Vitrobot Mark IV (Thermo Fisher). The grids were subsequently transferred to a Titan Krios (G3i) electron microscope (Thermo Fisher) operating at 300 kV and equipped with K3 direct electron detector (Gatan) and BioQuantum energy filter. The best grids were found from the sample at 1.5 mg/ml concentration.
A total of 14,094 and 8715 movies were automatically collected using the EPU Data Acquisition Software (Thermo Fisher) for non-tilted and 30-degree tilted collections, respectively. Both datasets were collected at a 0.86 Å pixel size and a dose of 50 e⁻/Å⁻² across 40 frames (0.0535 or 0.07025 sec per frame for the non-tilted and tilted data collection, respectively) at a dose rate of approximately 23.36 or 17.8 e⁻/Ų/s, respectively. A set of defocus values was applied ranging from −0.8, −1.0, −1.2, −1.4, or −1.8 μm with an energy filter slit width of 20 eV and a 100 μm objective aperture.