Cryo-EM Structure Determination of Cav2.3

Aliquots of 3.5 μl concentrated WT or ΔCH2 Ca_v2.3 proteins were loaded onto glow-discharged holey carbon grids (Quantifoil Cu/Au R1.2/1.3, 300 mesh), which were blotted for 6 s and plunge-frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher) at 8 °C with 100% humidity. Grids were transferred to a Titan Krios electron microscope (Thermo Fisher) operating at 300 kV and equipped with a Gatan Gif Quantum energy filter (slit width 20 eV) and spherical aberration (Cs) image corrector. Micrographs were recorded using a K2 Summit counting camera (Gatan) in super-resolution mode with a nominal magnification of 105,000×, resulting in a super-resolution pixel size of 0.557 Å. Each stack of 32 frames was exposed for 5.6 s, with an exposure time of 0.175 s per frame. The total dose for each stack was about 50 e⁻ per Å². The dose rate is 10.6 e^-/ pixel/s. SerialEM was used for fully automated data collection^{39 (link)}. All 32 frames in each stack were aligned, summed, and dose-weighted using MotionCorr2^{40 (link)} and twofold-binned to a pixel size of 1.114 Å per pixel. The defocus values were set from −1.9 to −2.1 μm and estimated by Gctf^{41 (link)}.

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Yao X., Wang Y., Wang Z., Fan X., Wu D., Huang J., Mueller A., Gao S., Hu M., Robinson C.V., Yu Y., Gao S, & Yan N. (2022). Structures of the R-type human Cav2.3 channel reveal conformational crosstalk of the intracellular segments. Nature Communications, 13, 7358.

Publication 2022

Vitrobot Mark IV Titan Krios electron microscope K2 Summit counting camera

Carbon Cav2 Ethane Frames Frozen Humidity Microscope electron Nitrogen Proteins

Corresponding Organization :

Other organizations : Princeton University, St. John's University, University of Oxford

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Variable analysis

independent variables

WT or ΔCH2 Ca_v2.3 proteins

dependent variables

Micrographs recorded using a K2 Summit counting camera in super-resolution mode

control variables

Glow-discharged holey carbon grids (Quantifoil Cu/Au R1.2/1.3, 300 mesh)
Blotting time of 6 s
Plunge-freezing in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV at 8 °C with 100% humidity
Titan Krios electron microscope operating at 300 kV with a Gatan Gif Quantum energy filter (slit width 20 eV) and spherical aberration (Cs) image corrector
Nominal magnification of 105,000×, resulting in a super-resolution pixel size of 0.557 Å
Exposure time of 0.175 s per frame, with a total dose of about 50 e^- per Å²
Dose rate of 10.6 e^-/pixel/s
Defocus values set from −1.9 to −2.1 μm

controls

No positive or negative controls were explicitly mentioned.

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