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Preparation of macromolecular complexes for cryo-electron microscopy

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Abstract

This protocol describes the preparation of frozen-hydrated single-particle specimens of macromolecular complexes. First, it describes how to create a grid surface coated with holey carbon by first inducing holes in a Formvar film to act as a template for the holey carbon that is stable under cryo-electron microscopy (cryo-EM) conditions and is sample-friendly. The protocol then describes the steps required to deposit the homogeneous sample on the grid and to plunge-freeze the grid into liquid ethane at the temperature of liquid nitrogen, so that it is suitable for cryo-EM visualization. It takes 4–5 h to make several hundred holey carbon grids and about 1 h to make the frozen-hydrated grids. The time required for sample purification varies from hours to days, depending on the sample and the specific procedure required. A companion protocol details how to collect cryo-EM data using an FEI Tecnai transmission electron microscope that can subsequently be processed to obtain a three-dimensional reconstruction of the macromolecular complex.

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Figure 1
Figure 2: Sample cryo-EM images.
Figure 3: Good holey Formvar.
Figure 4: Pseudo-holey Formvar.
Figure 5: Carbon-coated mica.
Figure 6: Micrograph of ribosomes in vitreous ice.
Figure 7: Manual plunge-freezing apparatus.

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Acknowledgements

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

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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. Wadsworth Center, Empire State Plaza, Albany, 12201-0509, New York, USA

    Derek J Taylor & Joachim Frank

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

    Joachim Frank

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  1. Robert A Grassucci
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Correspondence to Joachim Frank.

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Grassucci, R., Taylor, D. & Frank, J. Preparation of macromolecular complexes for cryo-electron microscopy. Nat Protoc 2, 3239–3246 (2007). https://doi.org/10.1038/nprot.2007.452

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