Holey carbon film

The purified NLGNs were used to prepare cryo-EM grids with a concentration of 0.25 mg ml⁻¹ and applied to the holey carbon film (Quantifoil, Großlöbichau, Germany; R1.2/1.3) grids. The grids were blotted for 2.5 s under 100% humidity at 4°C with Vitrobot Mark IV (Thermo Fisher) and plunge-frozen into pre-cooled liquid ethane. The grids were then observed using Titan Krios microscope (Thermo Scientific) operated at 300 kV and equipped with K3 Summit camera (Gatan, Pleasanton, CA, USA) for NLGN2 or K2 Summit camera (Gatan) for NLGN3. Micrographs were recorded with SerialEM under a nominal defocus value ranging from −1.5 to −2.5 μm and nominal magnification of ×130k for NLGN2 and ×165k for NLGN3, corresponding to calibrated pixel size of 0.92 Å pix⁻¹ for NLGN2 and 0.842 Å pix⁻¹ for NLGN3. A total of 4,021 and 7,984 micrographs were collected for NLGN2 and NLGN3, respectively. A detailed description of the data collection parameters is available in Supplementary Table 1.

Graphite powder (purity 99.9995%, 2–15 μm flakes, Alfa Aesar) was mixed into sulphuric acid, and then potassium permanganate and sodium nitrate added portion-wise. For the oxidation, water was added and the reaction mixture heated to 98 °C for 3 weeks. Terminating the reaction was followed by filtering, washing, and drying. To exfoliate the resulting graphite oxide powder into single-layer flakes, it was mixed with deionized water, vigorously stirred for 24 h, followed by bath sonication for 3 h, tip sonication for 30 min, and finally bath sonication for a further 1 h. To prepare the TEM samples, a Au support grid covered with a holey carbon film (Quantifoil) was dipped into a water-based dispersion for 1 min and then rinsed in isopropanol and dried in air^{52 (link)}.