Sample vitrification was carried out in an FEI Vitrobot plunge-freezing device set to an operating temperature of 10 °C with 90% relative humidity and 0 force for the blotting pad. Three microliters of the sample were applied to the freshly glow-discharged EM grid (company&size); after 180 s, the grid was blotted for 0.5 s and plunged into the liquid nitrogen-cooled liquid ethane to prepare the cryo-sample. It was subsequently cryo-transferred to a cryogenic holder cooled down to liquid-nitrogen temperature. The sample was then imaged in a field-emission transmission electron microscope (JEOL 2100F) operated at 200 keV in an annular dark-field scanning transmission electron microscopy mode (ADF-STEM). In this imaging mode, spatial resolution is not reduced by the inelastic scattering events underwent in the sample. In contrast, these inelastically scattered electrons would render significant resolution loss due to the chromatic aberration of the conventional TEM imaging mode. Therefore, ADF-STEM is superior to conventional TEM when imaging through micron-thick ices.
Vitrobot plunge freezing device
Manufactured by Thermo Fisher Scientific
The Vitrobot plunge-freezing device is a specialized piece of laboratory equipment used to rapidly freeze samples for various applications, such as cryo-electron microscopy. The device rapidly freezes samples in a controlled environment to preserve their native structure and minimize the formation of ice crystals. The core function of the Vitrobot is to provide a consistent and reproducible method for preparing samples for further analysis or imaging.
Automatically generated - may contain errors
3 protocols using vitrobot plunge freezing device
1
Cryo-EM Imaging of Samples in Thick Ice
2
Cryo-EM Structural Analysis of Kinesin-MT Complexes
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MTs were diluted in BrB20 to a final concentration of 5 μM. Kin1 and Kin3 were diluted in BrB20 containing either 2 mM of AMPPNP, ADP, ADP + AlF4, or apyrase (10 units/ml), according to established protocols (Hirose and Amos, 2007 (link); Sindelar and Downing, 2007 (link), 2010 (link); Fourniol and Moores, 2011 (link)), and warmed to room temperature 10 min prior to complex formation. The final concentrations used to visually achieve full decoration in the various nucleotide states are shown in Table 4 . C-flat holey carbon grids (Protochips, Raleigh, NC) with 2 μm holes and 4 μm spacing were glow-discharged in air. 4 μl drops of MT then Kin1 or Kin3 samples were added and blotted in sequential fashion using a Vitrobot plunge-freezing device (FEI Co., Hillsboro, OR) operating at 25°C and 100% humidity and vitrified in liquid ethane.10.7554/eLife.03680.026
Final protein concentrations used for cryo-EM sample preparation
| Kinesin and nucleotide state | [MT] (μM) | [Motor domain] (μM) |
|---|---|---|
| Kin3 MgADP | 5 | 10 |
| Kin3 NN | 5 | 5 |
| Kin3 Mg-AMPPNP | 5 | 5 |
| Kin3 Mg-ADP.AlFx | 5 | 5 |
| Kin1 NN | 5 | 100 |
| Kin1 Mg-AMPPNP | 5 | 50 |
| Kin1 Mg-ADP.AlFx | 5 | 50 |
Kin1 samples required higher concentrations than Kin3 to achieve good MT occupancy.
3
Cryo-EM Analysis of Chaperone-Usher Pili
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A 3 μL sample of type 1 chaperone-usher pili was applied to a glow-discharged Quantifoil 1.2/1.3 400 mesh grid (Agar Scientific) and incubated for 30 s before being blotted and plunged into liquid ethane using a Vitrobot plunge-freezing device (FEI). The data were collected on a Tecnai G2 Polara microscope (FEI) operated at 300 kV equipped with a K2 Summit direct electron detector (Gatan) operated in counting mode, placed at the end of a Quantum energy filter operated with a slit width of 20 eV, with a 1.13 Å pixel size and a defocus range of -0.5 to -3.5 μm. A total dose of 100 electrons/Å2 was applied and fractionated equally among 59 frames to allow for dose weighting.
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