Gatan cryo transfer holder model 626

The cryo-TEM
data were obtained at the BioCryo facility of the Northwestern University
Nuance Center (remote access due to COVID19 restrictions). The samples
were applied to 200 mesh Cu grids with a lacey carbon membrane. Before
plunge-freezing, grids (EMS Cat# LC200-CU-100) were glow discharged
in a Pelco easiGlow glow discharger (Ted Pella Inc., Redding, CA,
USA) using an atmosphere plasma generated at 15 mA for 15 s with a
pressure of 0.24 mbar. This treatment created a negative charge on
the carbon membrane, allowing liquid samples to spread evenly over
the grid. A 4 μL volume of each sample was pipetted onto the
grid and blotted for 5 s with a blot offset of +0.5 mm, followed by
immediate plunging into liquid ethane within an FEI Vitrobot Mark
III plunge freezing instrument (Thermo Fisher Scientific, Waltham,
MA, USA). Grids were then transferred to liquid nitrogen for storage.
The plunge-frozen grids were kept vitreous at −180 °C
in a Gatan Cryo Transfer Holder model 626.6 (Gatan Inc., Pleasanton,
CA, USA) while viewing using a Hitachi HT7700 W-emission transmission
electron microscope at 100 kV. Image data were collected by a Gatan
Orius 4k × 2.67k digital camera (Gatan Inc., Pleasanton, CA,
USA). Samples required no dilution at 1 wt % concentration for successful
visualization. Cryo-TEM images were analyzed using ImageJ software.

For cryo-TEM measurement, 200 mesh Cu grids with a lacey carbon membrane (EMS Cat. # LC200-CU) were placed in a Pelco easiGlow glow discharger (Ted Pella Inc., Redding, CA, USA) and an atmosphere plasma was introduced on the surface of the grids for 30 s with a current of 15 mA at a pressure of 0.24 mbar. This treatment creates a negative charge on the carbon membrane, allowing for aqueous liquid samples to spread evenly over of the grid. Then, 4 µL of sample was pipetted onto the grid and blotted for 5 s with a blot offset of +0.5 mm, followed by immediate plunging into liquid ethane within a FEI Vitrobot Mark III plunge freezing instrument (Thermo Fisher Scientific, Waltham, MA, USA). Grids were then transferred to liquid nitrogen for storage. The plunge-frozen grids were kept vitreous at –172 °C in a Gatan Cryo Transfer Holder model 626.6 (Gatan Inc., Pleasanton, CA, USA) while viewing in a JEOL JEM1230 LaB6 emission TEM (JEOL USA, Inc., Peabody, MA) at 120 keV. Image data were collected by a Gatan Orius SC1000 CCD camera Model 831 (Gatan Inc., Pleasanton, CA, USA). Image analysis was done using ImageJ.