1. Prepare sample and transport to electron microscopy facility on ice.
2. Select and glow-discharge electron microscopy grids.
3. Add fiducial markers to sample and vitrify immediately.
4. Screen grids on a screening electron cryo-microscope until desirable conditions are found. As there is inevitable variability between vitrified grids,
typically three or more identical grids will be frozen for subsequent data
collection.
4. Load specimen into microscope and collect tilt series:
1. Align and configure microscope (1 h).
2. Load grids into the aligned microscope suitable for data collection (1–2 h).
3. Configure data collection software with parameters appropriate to the
question (1 h).
4. Identify targets (1–5 h).
5. Acquire tilt series (15–120 min per tilt series, depending on data collection
parameters and acquisition software).
5. Reconstruct tomograms:
1. Transfer tilt series to a processing computer for reconstruction.
2. Reconstruct tomograms manually, semi-automatically, or fully-automatically.
6. Post-process tomogram
1. Filter and segment, or subtomogram average and generate isosurface.
7. Interpret data
8. Publish!
1. Write paper, submit, and manage peer review.
2. Archive data in EMDB and EMPIAR.
Acknowledgements The authors would like to thank Bonnie Chaban, Peter Rosenthal, and Louie
Henderson for insightful comments. This work has been supported by BBSRC grant
BB/L023091/1 to MB.
Bibliography
1. C.M. Oikonomou, G.J. Jensen, A new view into prokaryotic cell biology from electron
cryotomography. Nat. Rev. Microbiol. (2016)
2. L. Gan, G.J. Jensen, Electron tomography of cells. Q. Rev. Biophys. 45(01), 27–56 (2012)
3. J.L.S. Milne, S. Subramaniam, Cryo-electron tomography of bacteria: progress, challenges
and future prospects. Nat. Rev. Microbiol. 7(9), 666–675 (2009)
4. V. Lučić, A. Rigort, W. Baumeister, Cryo-electron tomography: the challenge of doing
structural biology in situ. J. Cell Biol. 202(3), 407–419 (2013)
88
J. L. Ferreira et al.
2. Select and glow-discharge electron microscopy grids.
3. Add fiducial markers to sample and vitrify immediately.
4. Screen grids on a screening electron cryo-microscope until desirable conditions are found. As there is inevitable variability between vitrified grids,
typically three or more identical grids will be frozen for subsequent data
collection.
4. Load specimen into microscope and collect tilt series:
1. Align and configure microscope (1 h).
2. Load grids into the aligned microscope suitable for data collection (1–2 h).
3. Configure data collection software with parameters appropriate to the
question (1 h).
4. Identify targets (1–5 h).
5. Acquire tilt series (15–120 min per tilt series, depending on data collection
parameters and acquisition software).
5. Reconstruct tomograms:
1. Transfer tilt series to a processing computer for reconstruction.
2. Reconstruct tomograms manually, semi-automatically, or fully-automatically.
6. Post-process tomogram
1. Filter and segment, or subtomogram average and generate isosurface.
7. Interpret data
8. Publish!
1. Write paper, submit, and manage peer review.
2. Archive data in EMDB and EMPIAR.
Acknowledgements The authors would like to thank Bonnie Chaban, Peter Rosenthal, and Louie
Henderson for insightful comments. This work has been supported by BBSRC grant
BB/L023091/1 to MB.
Bibliography
1. C.M. Oikonomou, G.J. Jensen, A new view into prokaryotic cell biology from electron
cryotomography. Nat. Rev. Microbiol. (2016)
2. L. Gan, G.J. Jensen, Electron tomography of cells. Q. Rev. Biophys. 45(01), 27–56 (2012)
3. J.L.S. Milne, S. Subramaniam, Cryo-electron tomography of bacteria: progress, challenges
and future prospects. Nat. Rev. Microbiol. 7(9), 666–675 (2009)
4. V. Lučić, A. Rigort, W. Baumeister, Cryo-electron tomography: the challenge of doing
structural biology in situ. J. Cell Biol. 202(3), 407–419 (2013)
88
J. L. Ferreira et al.