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

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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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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, 10032, New York, USA

    Robert A Grassucci & Joachim Frank

  2. Health Research Incorporated at the Wadsworth Center, Empire State Plaza, Albany, 12201-0509, New York, USA

    Derek Taylor

  3. Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, New York, 10027, New York, USA

    Joachim Frank

Authors
  1. Robert A Grassucci
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  2. Derek Taylor
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  3. Joachim Frank
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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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