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.

Figure 1

Schematic representation of grid preparation steps.

Figure 2

Sample cryo-EM images. (a) Ethane contamination. The high-contrast blobs are contaminants that are sometimes in the ethane used to plunge-freeze the grids. The scale bar represents 250 nm on the object scale. (b) Low-magnification cryo-EM image of a bad grid. The image shows various problems that can occur when making frozen-hydrated grids. BC indicates large ice contaminants that are usually the result of poor handling. SP indicates the ‘splotchy’ ice appearance that occurs when the substrate is not hydrophilic. TI indicates thick ice. The scale bar is 2 µm on the object scale. (c) Vitreous and crystalline ice. Cryo-electron micrograph showing the appearance of vitreous ice (V) and crystalline ice (X). The scale bar represents 250 nm on the object scale. (d) Freeze-dried particles. Cryo-electron micrograph showing the high-contrast appearance of particles that have been freeze-dried. The scale bar represents 250 nm on the object scale.

Figure 3

Good holey Formvar. Image from phase-contrast LM showing the appearance of a good network of holes in a Formvar-coating on a glass slide.

Figure 4

Pseudo-holey Formvar. Image from a phase-contrast LM showing the appearance when the Formvar coating is not fully perforated.

Figure 5

Carbon-coated mica. A thin layer (approximately 50 Å) of carbon is deposited onto freshly cleaved mica using a vacuum evaporator. The thickness of the carbon can be estimated by monitoring the gray level on the filter paper (see red arrow). Also note the voids left by the holey carbon grids in the center of the image that were recoated along with the mica.

Figure 6

Micrograph of ribosomes in vitreous ice. The micrograph shows an even particle distribution of ribosomes on a frozen-hydrated grid. Note: Contrast has been inverted for subsequent image processing.

Figure 7

Manual plunge-freezing apparatus. The sample is applied to the grid held in place by the forceps (blue arrow). The sample layer is blotted between the filter pads (green arrow) and quickly plunged into the cryogen cup (red arrow).