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Preparation of macromolecular complexes for cryo-electron microscopy

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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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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