Cryo-EM Sample Preparation for RNC Imaging

Approximately 3 μL of freshly made RNC at a concentration of 40 to 60 nM were incubated for 1 min on plasma-cleaned 300-mesh holey carbon grids (C-flat R2/2), on which a home-made continuous carbon film was pre-deposited. Grids were blotted for 4 seconds with 100% humidity at 4 °C and plunge-frozen in liquid ethane using an FEI Vitrobot. Automated data collection was performed on a Titan Krios electron microscope (FEI) equipped with a K2 Summit direct detector and GIF Quantum filter (Gatan) at 300 kV (Tables 1-4). The total exposure time was 9 seconds, with a total dose of 50 electrons per Å² (frame dose 1.3 electrons per Å²) ^{39 (link)}.

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Li W., Ward F.R., McClure K.F., Tsai-Lan Chang S., Montabana E., Liras S., Dullea R.G, & Cate J.H. (2019). Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule. Nature structural & molecular biology, 26(6), 501-509.

Publication 2019

Vitrobot Titan Krios electron microscope K2 Summit direct GIF Quantum filter

Carbon Electrons Ethane Frame Frozen Humidity Microscope electron Plasma Seconds

Corresponding Organization :

Other organizations : University of California, Berkeley, Pfizer (United States), Lawrence Berkeley National Laboratory

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Variable analysis

independent variables

Incubation time of RNC on plasma-cleaned 300-mesh holey carbon grids (1 min)

dependent variables

Cryo-electron microscopy data collection

control variables

Concentration of freshly made RNC (40 to 60 nM)
Type of grids used (plasma-cleaned 300-mesh holey carbon grids with pre-deposited home-made continuous carbon film)
Blotting time (4 seconds)
Blotting humidity (100%)
Blotting temperature (4 °C)
Plunge-freezing in liquid ethane using an FEI Vitrobot
Electron microscope used (Titan Krios at 300 kV)
Detector used (K2 Summit direct detector)
Energy filter used (GIF Quantum filter)
Total exposure time (9 seconds)
Total dose (50 electrons per Å^2)
Frame dose (1.3 electrons per Å^2)

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