L. pneumophila Lp02 cells were grown as mentioned previously12 (link), harvested at early stationary phase (OD600 of ~3.0), mixed with 10-nm colloidal gold beads (Sigma-Aldrich, St. Louis, MO) precoated with bovine serum albumin, and applied onto freshly glow-discharged copper R2/2 200 Quantifoil holey carbon grids (Quantifoil Micro Tools GmbH, Jena, Germany). Grids were then blotted and plunge-frozen in a liquid ethane/propane mixture35 (link) using an FEI Vitrobot Mark IV and stored in liquid nitrogen for subsequent imaging.
Protocol detail
Cryo-EM sample preparation for Legionella
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Corresponding Organization : Howard Hughes Medical Institute
Other organizations : Washington University in St. Louis, University of Florida, Scripps Research Institute
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Variable analysis
independent variables
- Growth conditions of
L. pneumophila Lp02 cells (as mentioned previously in reference 12)
- Harvesting of
L. pneumophila Lp02 cells at early stationary phase (OD600 of ~3.0) - Mixing of
L. pneumophila Lp02 cells with 10-nm colloidal gold beads precoated with bovine serum albumin - Application of the mixture onto freshly glow-discharged copper R2/2 200 Quantifoil holey carbon grids
- Blotting and plunge-freezing of the grids in a liquid ethane/propane mixture using an FEI Vitrobot Mark IV
- Subsequent imaging of the frozen grids
- Quantifoil holey carbon grids (copper R2/2 200)
- 10-nm colloidal gold beads precoated with bovine serum albumin
- Liquid ethane/propane mixture for plunge-freezing
- FEI Vitrobot Mark IV for plunge-freezing
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