Krios g3i microscope

Purified 8C-8A(IL1²⁵) was applied to 300 mesh UltrAuFoil holey gold 1.2/1.3 grids (Quantifoil Microtools) that were glow discharged for 10 s at 25 mA. The grids were blotted for 4 s at force 12 using double-layer Whatman filter papers (1442-005, GE Healthcare) before plunging into liquid ethane using an FEI MarkIV Vitrobot at 8°C and 100% humidity. Samples were imaged using a 300 kV FEI Krios G3i microscope equipped with a Gatan K3 direct electron camera. Movies containing 400 frames were collected in super-resolution mode at ×81,000 magnification with a physical pixel size of 1.1 Å/pixel and defocus values at a range of –0.8 to –1.5 µm using the automated imaging software SerialEM (Mastronarde, 2005 (link)).
8C-8A(IL1²⁵) nanodisc grids were prepared as described above. Samples were imaged using a 300 kV FEI Krios G4 microscope equipped with a Gatan K3 direct electron camera. Movies containing 50 frames were collected in super-resolution mode at ×103,000 magnification with a physical pixel size of 0.818 Å/pixel and defocus values at a range of –0.8 to –2.2 µm using the automated imaging software EPU (Thermo Fisher Scientific).

Purified hIP₃R-3 in the SEC buffer containing 1 mM EDTA was supplemented with 500 µM IP₃ (from 10 mM stock in water), 0.1 mM CaCl₂, and 5 mM ATP (from 100 mM stock, pH 7.2). 2.0 μL of the protein sample was applied to 300 mesh Cu Quantifoil 1.2/1.3 grids (Quantifoil Microtools) that were glow discharged for 20 s at 25 mA. The grids were blotted for 7 s at force 10 using single-layer Whatman ashless filter papers (Cat. #: 1442-055, GE Healthcare) and were plunged into liquid ethane using an FEI MarkIV Vitrobot at 8 °C and 100% humidity. The filter papers were not pre-treated with Ca²⁺ chelators or any other chemicals. Four grids prepared using the same sample were imaged using a 300 kV FEI Krios G3i microscope equipped with a Gatan K3 direct electron camera in four different data collection sessions at Case Western Reserve University. Movies containing 40–50 frames were collected at a magnification of ×105,000 in super-resolution mode with a physical pixel size of 0.828 Å/pixel and defocus values at a range of −0.8 to −1.6 µm using the automated imaging software SerialEM^{47 (link)} and EPU (ThermoFisher Scientific).

The vitrified samples of the G. lamblia NCP were imaged at 300 kV on a Krios G3i microscope (Thermo Fisher Scientific), equipped with an energy-filtered K3 detector calibrated at a pixel size of 1.05 Å with a slit width of 25 eV, using the SerialEM auto acquisition software (34 (link)). The images of the G. lamblia NCP were recorded with a 5.6 second exposure time in the electron counting mode with a 140 milliseconds frame time, retaining a stack of 40 frames with an accumulated total dose of ∼64 electrons/Ų.