Tecnai g2 spirit microscope

Negative stain was performed according to standard protocols. Samples in buffer were applied to glow-discharged carbon-coated copper grids. Excess sample was removed by gently blotting the grid edge with filter paper after 40 seconds. The grid was sequentially washed in two drops of deionized water with intermittent blotting steps. The grid was then briefly dipped in a first drop of 0.75% uranyl formate, blotted, and then stained for 30 seconds in a second drop. The grid was air dried after a final blotting step. Micrographs were collected on a FEI Tecnai G2 Spirit microscope.

20 µM PfActI was polymerized with or without 25 µM CPs in F-buffer at 20 °C for 16 h. Prior sample application, the carbon-coated 300-mesh copper grids (Gilder, US) were glow-discharged for 1 min. After diluting PfActI to 1 µM with F-buffer, grids were immediately prepared using a previously reported protocol [8 (link)]. Negative stained grids were imaged using a Tecnai G2 Spirit microscope (FEI, US) operated at 120 kV with a pixel size of 0.592 nm. Micrographs were analyzed using the Ridge Detection plugin [92 ] of Fiji [93 (link)]. Particles smaller than 12 nm were excluded to omit bias caused by any unbound CP. Mann–Whitney tests were carried out using OriginPro (OriginLab, US).

SEC fractions containing hTie2 Ig3–Fn3 D682C/N691C in complex with hTAAB IgG1 were diluted to 0.01 mg/ml in 20 mM HEPES, pH 7.5, 200 mM NaCl, and 2.5 μl of the protein was placed onto a glow-discharged CF300-CU grid (Electron Microscopy Sciences). After a 30-s incubation, the sample was blotted with Whatman paper, dipped two times into 20 μl of distilled water, and stained twice with 20 μl of a 0.76% uranyl formate solution for 1 min. Electron micrographs were collected at ×52,000 magnification on a Tecnai G2 spirit microscope (FEI) at 120 kV with a 4 K × 4 K Eagle camera (FEI), using a −1.8 μm defocus value and a dose of 30 e⁻/Ų. The resulting pixel size was 2.11 Å per pixel. Micrographs were imported into cryoSPARC^{55 (link)}, and contrast transfer function parameters were calculated using patch CTF estimation. A total of 2579 particles were manually selected from 38 micrographs, and 2D class averages were generated after extracting the particles with a 320-pixel box and binning to 160 pixels. 2D classification was run in cryoSPARC with default settings, except the number of 2D classes was set to 8. Objectively good (showing clear Fc and Tie2 density) class averages were selected for further analysis.

Transmission electron microscopy (TEM) measurements were done at the Tecnai G2 Spirit microscope from FEI operating at 120 kV, and high-resolution scanning TEM (HRSTEM) measurements were carried out on JEOL NEOARM 200 F operating at 200 kV equipped with Schottky-FEG cathode and C_S corrector, respectively. In both techniques, the toluene dispersion of NPs was dropped at the Cu grid with 400 mesh coated by carbon foil. The TEM micrographs were taken in bright-field mode, and the size distribution was made by manually measuring at least 200 particle sizes from different TEM micrographs in ImageJ software.^{67 (link)} Acquisition of HRSTEM micrographs was made in annular bright (ABF) and dark-field (ADF) mode.