Imaging
was carried out using a field emission cryo-electron microscope
(JEOL JEM-3200FSC), operating at 200 kV. Images were taken in bright
field mode and using zero loss energy filtering (omega type) with
a slit width of 20 eV. Micrographs were recorded using a Gatan Ultrascan
4000 CCD camera. The specimen temperature was maintained at −187
°C during the imaging. Vitrified specimens were prepared using
an automated FEI Vitrobot device using Quantifoil 3.5/1 holey carbon
copper grids with a hole size of 3.5 μm. Just prior to use,
grids were plasma cleaned using a Gatan Solarus 9500 plasma cleaner
and then transferred into the environmental chamber of a FEI Vitrobot
at room temperature and 100% humidity. Thereafter, 3 μL of the
sample solution was applied on the grid and it was blotted twice for
5 s and then vitrified in a 1:1 mixture of liquid ethane and propane
at a temperature of −180 °C. The grids with vitrified
sample solution were maintained at liquid nitrogen temperature and
then cryo-transferred to the microscope.
was carried out using a field emission cryo-electron microscope
(JEOL JEM-3200FSC), operating at 200 kV. Images were taken in bright
field mode and using zero loss energy filtering (omega type) with
a slit width of 20 eV. Micrographs were recorded using a Gatan Ultrascan
4000 CCD camera. The specimen temperature was maintained at −187
°C during the imaging. Vitrified specimens were prepared using
an automated FEI Vitrobot device using Quantifoil 3.5/1 holey carbon
copper grids with a hole size of 3.5 μm. Just prior to use,
grids were plasma cleaned using a Gatan Solarus 9500 plasma cleaner
and then transferred into the environmental chamber of a FEI Vitrobot
at room temperature and 100% humidity. Thereafter, 3 μL of the
sample solution was applied on the grid and it was blotted twice for
5 s and then vitrified in a 1:1 mixture of liquid ethane and propane
at a temperature of −180 °C. The grids with vitrified
sample solution were maintained at liquid nitrogen temperature and
then cryo-transferred to the microscope.