Cryo-EM Sample Preparation Protocol

The cryo-EM sample preparation followed our previous publication (Benecke et al., 2022 (link)). A total of 4 μL aliquot of each sample was adsorbed onto holey carbon-coated grid (Ted Pella Inc., Redding, United States) blotted with Whatman 1 filter paper and vitrified into liquid ethane at −178°C using a Leica GP plunger (Leica Microsystems GmbH, Wetzlar, Germany). Frozen grids were transferred onto a Talos electron microscope (FEI/Thermo Fisher Scientific Inc., Waltham, United States) using a Gatan 626 cryo-holder (Gatan, Inc., Pleasanton, United States). Electron micrographs were recorded at an accelerating voltage of 200 kV, using a low-dose system (20 e−/Å²) and keeping the sample at low temperature. Micrographs were recorded on a CETA camera (Thermo Fisher Scientific Inc., Waltham, United States).

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Yu M.S., Chiang D.M., Reithmair M., Meidert A., Brandes F., Schelling G., Ludwig C., Meng C., Kirchner B., Zenner C., Muller L, & Pfaffl M.W. (2024). The proteome of bacterial membrane vesicles in Escherichia coli—a time course comparison study in two different media. Frontiers in Microbiology, 15, 1361270.

Publication 2024

holey carbon-coated grid Whatman 1 filter paper Leica GP plunger Gatan 626 cryo-holder CETA camera

Corresponding Organization :

Other organizations : Technical University of Munich, Ludwig-Maximilians-Universität München, University of Basel, LMU Klinikum, Leibniz-Institute for Food Systems Biology at the Technical University of Munich, University Hospital of Basel

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Variable analysis

independent variables

Sample preparation protocol (Benecke et al., 2022)
Aliquot volume (4 μL)
Grid type (holey carbon-coated)
Blotting (Whatman 1 filter paper)
Vitrification method (liquid ethane at −178°C using a Leica GP plunger)
Electron microscope type (Talos)
Accelerating voltage (200 kV)
Electron dose (20 e−/Å^2)

dependent variables

Electron micrographs

control variables

Sample temperature (low temperature)
Cryo-holder (Gatan 626)
Camera type (CETA)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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