Cryo-EM Imaging of 2D Protein Crystals

3.5 µl of solution containing 2D crystals were applied to glow-discharged Quantifoil R1.2/1.3 grids for 1 min, blotted for 3 s and then vitrified by plunging into liquid nitrogen-cooled liquid ethane in a FEI Vitrobot Mark IV (FEI). Samples were transferred to an FEI Titan Krios and 2D crystals were imaged for 2 s in 100 ms frames at a dose of 1 electron per Å per second at 22,500x in super-resolution counting mode using a Gatan K3 direct electron detector. Images were corrected for drift using whole frame and patch algorithms and Fourier cropped using MotionCorr2^{66 (link)}. Images were unbent and the best 8 images were merged using a lattice of a = b = 95 Å and γ = 90˚ using Focus^{67 (link)}. The best 8 images based on merging phase residual were merged to calculate a projection map in layer group p42₁2 with a 4 Å resolution limit.

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Jiang Y., Thienpont B., Sapuru V., Hite R.K., Dittman J.S., Sturgis J.N, & Scheuring S. (2022). Membrane-mediated protein interactions drive membrane protein organization. Nature Communications, 13, 7373.

Publication 2022

R1.2/1.3 grids Vitrobot Mark IV Titan Krios

Electron Ethane Frame Nitrogen

Corresponding Organization : Cornell University

Other organizations : Aix-Marseille Université, Centre National de la Recherche Scientifique, Memorial Sloan Kettering Cancer Center

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Variable analysis

independent variables

Amount of solution containing 2D crystals applied to grids (3.5 µl)
Blotting time (3 s)
Imaging time (2 s)
Electron dose (1 electron per Å per second)

dependent variables

Images of 2D crystals

control variables

Glow-discharged Quantifoil R1.2/1.3 grids
Vitrification by plunging into liquid nitrogen-cooled liquid ethane in a FEI Vitrobot Mark IV
Imaging on an FEI Titan Krios at 22,500x in super-resolution counting mode using a Gatan K3 direct electron detector
Image drift correction using whole frame and patch algorithms
Fourier cropping using MotionCorr2
Image unbending and merging using Focus to calculate a projection map in layer group p42(1)2 with a 4 Å resolution limit

controls

No explicit positive or negative controls mentioned.

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