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Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes

Abstract

This protocol details the steps used for visualizing the frozen-hydrated grids as prepared following the accompanying protocol entitled 'Preparation of macromolecular complexes for visualization using cryo-electron microscopy.' This protocol describes how to transfer the grid to the microscope using a standard cryo-transfer holder or, alternatively, using a cryo-cartridge loading system, and how to collect low-dose data using an FEI Tecnai transmission electron microscope. This protocol also summarizes and compares the various options that are available in data collection for three-dimensional (3D) single-particle reconstruction. These options include microscope settings, choice of detectors and data collection strategies both in situations where a 3D reference is available and in the absence of such a reference (random-conical and common lines).

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Figure 1
Figure 2: Cryo-transfer system overview.
Figure 3: Cryo-transfer station.
Figure 4
Figure 5: Tools and cartridges.
Figure 6
Figure 7: The transfer station.
Figure 8: Transfer vessel with MSC inserted.
Figure 9: Turbo.
Figure 10: Diagram of grid square showing various low-dose areas.
Figure 11: Low-dose UI.
Figure 12: Micrograph of ribosomes in vitreous ice.

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Acknowledgements

We acknowledge the support from HHMI, NIH R37 GM29169, R01 GM55440 and P41 RR01219. We also thank M. Watters for assistance in generating figures.

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Correspondence to Joachim Frank.

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Grassucci, R., Taylor, D. & Frank, J. Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes. Nat Protoc 3, 330–339 (2008). https://doi.org/10.1038/nprot.2007.474

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  • DOI: https://doi.org/10.1038/nprot.2007.474

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