Cryoarm300

For dimeric human α-catenin (residues 22–906) bound to F-actin, grids were prepared by reconstituting the complex on the grid. C-flat 1.2/1.3 400 mesh grids were glow discharged for 60 s and placed into a Leica GP2 cryogenic plunger set to 65% humidity and 21 °C. First, 3.5 μl of 0.7 μM F-actin diluted in 150 NaCl, 20 mM Tris pH 7.5, and 1 mM dithiothreitol was placed on the carbon film side of the C-flat grid and left to incubate for 1 min before the successive addition and removal of 3.5 μl of 7.6 μM (0.74 mg/ml) F-actin. The addition and removal of α-catenin was repeated four times before blotting from the backside of the grid. Grids were immediately plunged frozen in liquid ethane and transferred into a JEOL cryoARM300 for screening and data collection.
Vitrification of human monomeric α-catenin was carried out by applying 3 μl of 0.25 mg/ml α-catenin on a glow discharged 300 mesh Au-Flat holey grids (ProtoChips) with 1.3 μm holes at 95% humidity and 4 °C using a Leica GP2 plunge freezer. The blotting was carried out for 6 seconds, and the grids were immediately plunged frozen into liquid ethane. Frozen grids were then transferred into a JEOL cryoARM300 for screening and data collection.

For the HMP1/α-catenin FABD in complex with F-actin cryoEM structure determination, C-Flat 1.2/1.3400 mesh grids were glow discharged for 60 s at 15 mA using a PELCO easiGlow (Ted Pella Inc) and mounted on Leica GP2 cryogenic plunger (65% humidity and 21 °C). First, 3.5 μl of F-actin (0.7 μM) was placed on the carbon film and incubated for 60 s and subsequently successive addition and removal of 3.5 μl of 0.6 mg/ml (21 μM) HMP1/α-catenin. The addition and removal steps were repeated twice before blotting from the backside of the grid for a total of 12 s. The grids were immediately plunge frozen in liquid ethane that is maintained at -183 K and cooled by liquid nitrogen. Lower concentrations of the HMP1/α-catenin FABD were unsuitable as they resulted in partial decoration. The grids were transferred into a Japan Electron Optics Laboratory (JEOL) cryoARM300 for screening and data collection.
For the full-length HMP1/α-catenin cryoEM structure determination, an Au-Flat 1.2/1.3300 mesh grid (Protochips) was glow discharged at 15 mA for 240 s using a PELCO easiGlow (Ted Pella Inc) and taken into Leica GP2 cryogenic plunger set to 95% humidity and 4 °C. About 4 μl of 0.3 mg/ml of full-length HMP1/α-catenin was applied on the carbon film side, blotted on the carbon side for 7 s, and plunged immediately for vitrification in liquid ethane and transferred into a JEOL cryoARM300.

Cryo-EM images were collected on a JEOL CryoARM 300 microscope equipped with an in-column Ω energy filter (Fislage et al., 2020 (link)) at 300 kV, automatically using SerialEM 3.0.8 (Mastronarde, 2005 (link)). The energy filter slit was set to 20 eV width. The nanodisc sample was collected at a nominal magnification of 60,000 and the corresponding calibrated pixel size of 0.771 Å. Five images per single stage position were collected using a cross pattern with three holes along each axis (Efremov and Stroobants, 2021 (link)). The 3 s exposures were dose-fractionated into 61 frames with an electron dose of 1.06 e- Å⁻² per frame. In total, 9122 zero-loss micrographs were recorded with the defocus varying between −0.9 and −2.2 µm (Table 1).
The LMNG-solubilized sample was collected at a 60,000 nominal magnification and the calibrated pixel size of 0.766 Å. Nine images were collected per stage position using a 3x3 hole pattern. The 3 s exposures were dose-fractionated into 60 frames with 1 e^- A⁻² dose per frame. The defocus varied between −1.0 and −2.0 µm. During 36 hr of data collection, 13,084 zero-loss micrographs were recorded.