Tecnai g2 sprit twin transmission electron microscope

The surface morphology and size of the BC-HDD NPs were analyzed using Transmission Electron Microscopy (TEM). Briefly, aqueous solution of freeze dried nanoparticles was prepared at the concentration of 1 mg/ml. A drop of this solution was placed on a TEM grid surface followed by the addition of a drop of 1% uranyl acetate onto the grid surface. After 1 min of incubation, excess fluid was removed and the grid surface was air dried at 25±2°C before loading onto the microscope. Then the NPs were observed under an electron microscope (FEI Tecnai G2 sprit Twin Transmission Electron Microscope equipped with CCD Camera, Netherlands) at 80 kV.

BN-PAGE-extracted YaxAB-C₈ pores were applied to glow-discharged, carbon-coated grids (carbon film 400 mesh Cu, Electron Microscopy Sciences, Hatfield, PA, USA) and stained using 0.8% (w/v) Uranyl Formate solution (Electron Microscopy Sciences, Hatfield, PA, USA). Images were acquired at 67,000-fold magnification on a TECNAI G2 Sprit TWIN transmission electron microscope (FEI Company, Hillsboro, OR, USA) operated at 120 kV using an Eagle 4 K High Sensitivity CCD detector (FEI company, Hillsboro, OR, USA). Automated particle selection and 2D classification were performed using CryoSPARC. The initial model of YaxAB-C₈ for the negative stain 3D reconstruction was generated from three representative classes of 2D averages with C₈ symmetry, and 5,000 particles were selected for 3D reconstruction. Based on a set of YaxAB heterodimers from the YaxAB-C₁₀ model (PDB code: “6EL1”), we generated the full model structure of YaxAB-C₈ through the rigid body fitting using Coot software^{55 (link)}. The model was subjected to several rounds of geometry minimization using phenix.geometry minimization, including the secondary structure and NCS restraints.