Cf200 cu ul

Cryo Transmission Electron Microscopy (TEM) samples were flash frozen onto lacey carbon film grids (LC200-CU, Electron Microscopy Sciences, Hatflield, PA, USA) and imaged on a FEI Talos TEM (FEI, Hillsboro, OR, USA) at an acceleration voltage of 200kV. Negative-stained samples were deposited on carbon coated square grids (CF200-Cu-UL, Electron Microscopy Sciences), dried, and stained with 2% uranyl formate, and imaged on a FEI Tecnai G2 Spirit TEM at an acceleration voltage of 120 kV.

The cryo-EM specimens were prepared following the procedure described before^{37 (link)}. Briefly, an aliquot (~3 μl) of nucleosome array sample ~400 nM was placed onto the 200 mesh Quantifoil copper grid (Q210CR-06, Electron Microscopy Sciences) that had been glow-discharged for ~15 s by (PELCO easiGlow^™ Glow Discharge Cleaning System) for 15 seconds. After incubating for ~10 sec, the grid was flash-frozen in liquid ethane at ~90% humidity and 4 °C with a Leica EM GP rapid-plunging device (Leica, Buffalo Grove, IL, USA) after being blotted with filter paper with a controlled blotting time (2 s). The flash-frozen grids were transferred into liquid nitrogen for storage.
The NS-EM specimens of nucleosome array sample were prepared using the optimized negative-staining protocol (OpNS) as described ^{67 (link)}. In brief, the samples were diluted to ~20 nM with sample buffer. An aliquot (~4 μL) of diluted sample was placed on an ultra-thin carbon-coated 200-mesh copper grid (CF200-Cu-UL, Electron Microscopy Sciences, Hatfield, PA, USA) that had been glow-discharged for ~15 s. After ~1 min incubation, the excess solution on the grid was blotted with filter paper. The grid was then washed with water and stained with 1% (w/v) uranyl formate (UF) before air-drying with nitrogen.

The OpNS specimens of IgG hole-hole homodimers and the NISTmAb were prepared by using the protocol as described^{35 (link)–38 (link)}. In brief, IgG samples were diluted to ~0.04 μg mL⁻¹ with Dulbecco’s phosphate-buffered saline (DPBS). An aliquot (approximately 4 μL) of diluted sample was placed on an ultra-thin carbon-coated 200-mesh copper grid (CF200-Cu-UL, Electron Microscopy Sciences, Hatfield, PA, USA, and Cu-200CN, Pacific Grid-Tech, San Francisco, CA, USA) that had been glow-discharged for 15 s. After 1-min incubation, the excess solution on the grid was blotted with filter paper. The grid was then washed with water and stained with 1% (w/v) uranyl formate (UF) before air-drying with nitrogen.

The NS-EM specimens of nucleosome array sample were prepared as described in a previously published protocol⁵⁹ . In brief, the samples were diluted to ~20 nM with sample buffer. An aliquot (~4 μL) of diluted sample was placed on an ultra-thin carbon-coated 200-mesh copper grid (CF200-Cu-UL, Electron Microscopy Sciences, Hatfield, PA, USA) that had been glow-discharged for ~15 s. After ~1 min incubation, the excess solution on the grid was blotted with filter paper. The grid was then washed with water and stained with 1% (w/v) uranyl formate (UF) before air-drying with nitrogen. For the time series incubation experiment, tetranucleosome array samples were diluted to 30 nM in 20 mM HEPES buffer under three different salt conditions (5 mM NaCl, 50 mM NaCl, and 150 mM NaCl). At the specified time point (2 min, 20 min, 60 min, 2.5 h, 5 h, and 10 h), 4 μL of the sample was used to prepare the NS-EM sample described above for each of the incubation solutions.