400 mesh copper grid

Carbon Film, 400 Mesh copper grids (Electron Microscopy Sciences) were glow discharged and exocyst holocomplex (1.9 μM) was applied for 10 minutes and blotted away with Whatman paper. Grids where washed twice with ddH₂O before being stained with 0.75% uranyl formate for approximately 30s. Excess stain was removed by blotting, and samples were imaged using a JEOL 2200FS 200 kV with inline omega filter on a Gatan Ultrascan 4k 15 μm pixel camera at 30,000x magnification. Images were obtained at a defocus of -0.5-2.0 μm. Particles were manually selected using Boxer implemented in EMAN2.⁷⁹ (link) Morphological model was generated in EMAN2 from 7052 particles over 75 micrographs to assess protein complex heterogeneity and dimensions.

Negative staining of the VRF was performed as described before (101 (link), 102 (link)). Briefly, 5 µL of VFF or VRF was adsorbed for 20 min at RT onto enhanced hydrophilicity-400 mesh copper grids (Electron Microscopy Sciences, PA, USA). The grids were then washed with PBS and ddH₂O, contrasted with 2% uranyl acetate at pH 4.4, air-dried, and then analyzed by transmission electron microscopy (Morgani 268 at 80 kV; FEI, Eindhoven, The Netherlands).

Adenine (1 mg/mL) was dissolved in PBS by heating to 90 °C for 3 h to obtain a monomeric solution. The adenine solution was then mixed with the chemical chaperones at the indicated concentrations. Adenine diluted in PBS served as a control for the osmolyte-treated samples, while 0.005% DMSO was supplemented as a control for samples treated with hydrophobic compounds. The solutions were gradually cooled down and incubated overnight at room temperature to facilitate self-assembly. Subsequently, the samples were vortexed and 10 µL of each was placed on 400-mesh copper grids (Electron Microscopy Sciences, Hatfield, PA, USA). The grids were incubated at room temperature for two minutes, and excess fluids were absorbed. Samples were visualized using a JEOL 1200EX electron microscope operating at 80 kV.

Four microliters of the cross-linked TRF1_core-teloNCP complex from the gradient was applied onto 400-mesh copper grids (Electron Microscopy Sciences, catalog no. G400-Cu) coated with a layer of homemade carbon film on nitrocellulose, which had been glow-discharged for 15 s at 30 mA with a Sputter Coater discharger (Edwards S150B). Following 1.5-min incubation, the grid was incubated with 2% (w/v) uranyl format for a total of 1 min. Data collection was performed using EPU (Thermo Fisher Scientific) on a 200-kV F20 Technai transmission electron microscope equipped with a Falcon II direct electron detector in linear mode with a physical pixel size of 2.02 Å/pixel and with a total dose of 66 electron/Ų over an exposure time of 1.49 s. A dataset of 788 micrographs was collected.

Common chemicals were purchased from Fisher Scientific and Sigma-Aldrich. Porcine brain tubulin, GTP and paclitaxel were purchased from Cytoskeleton, Inc. 99.8% D₂O, ¹⁵NH₄Cl and U-¹³C₆ glucose, 2-¹³C glucose, 1,6-¹³C glucose, U-¹³C₆,D₇ glucose were purchased from Cambridge Laboratories, Inc. EDTA-free protease inhibitor tablets were obtained from Roche. Chromatography columns were purchased from GE Healthcare. The 400 mesh copper grids coated with formvar and stabilized with evaporated carbon films were purchased from Electron Microscopy Science.
The human KIF5B motor domain construct (amino acids 1–349) was prepared using the pET28b vector fused with a His₆-SMT3 Tag at the N-terminus (in the form of His₆-SMT3-KIF5B). The His₆-SMT3-KIF5B was transformed into Escherichia coli BL21(DE3) cells. The His₆-Ulp1 was expressed and purified from the His₆-Ulp1 protease construct kindly provided by Dr. Christopher Lima from HHMI and Memorial Sloan Kettering Cancer Center, New York, NY 10065.