Protocol detail

Cryo-EM Imaging of Vitrified Samples

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Sample preparation was as described (Monroe et al., 2017 (link)). Vitrified grids were loaded onto a Titan Krios (FEI) operating at 300 kV. Images were acquired using a defocus range between −1.0 to −2.2 µm. A total of 2,349 cryo-EM movies were recorded using a K2 Summit direct detector (Gatan) in counting mode with a pixel size of 1.10 Å and at a dose rate of ~7.4 e^-/pixel/sec. Each movie was recorded as a stack of 40 frames accumulated over 10 s, totaling ~62e^-/Å².

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Han H., Monroe N., Sundquist W.I., Shen P.S, & Hill C.P. (2017). The AAA ATPase Vps4 binds ESCRT-III substrates through a repeating array of dipeptide-binding pockets. eLife, 6, e31324.

Publication 2017

Titan Krios K2 Summit direct detector

Frames

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Variable analysis

independent variables

Voltage of Titan Krios electron microscope (300 kV)

dependent variables

Defocus range (-1.0 to -2.2 µm)
Number of cryo-EM movies recorded (2,349)
Pixel size of K2 Summit direct detector (1.10 Å)
Dose rate of electron beam (~7.4 e-/pixel/sec)
Total electron dose per movie (~62 e-/Å2)

control variables

Sample preparation as described in Monroe et al., 2017

Annotations

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