Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

Summary

This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).

Abstract

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large macromolecular complexes. Compared to X-ray crystallography, it has some unique advantages. First, single particle EM reconstruction does not need to crystallize the protein sample, which is the bottleneck in X-ray crystallography, especially for large macromolecular complexes. Secondly, it does not need large amounts of protein samples. Compared with milligrams of proteins necessary for crystallization, single particle EM reconstruction only needs several micro-liters of protein solution at nano-molar concentrations, using the negative staining EM method. However, despite a few macromolecular assemblies with high symmetry, single particle EM is limited at relatively low resolution (lower than 1 nm resolution) for many specimens especially those without symmetry. This technique is also limited by the size of the molecules under study, i.e. 100 kDa for negatively stained specimens and 300 kDa for frozen-hydrated specimens in general.

For a new sample of unknown structure, we generally use a heavy metal solution to embed the molecules by negative staining. The specimen is then examined in a transmission electron microscope to take two-dimensional (2D) micrographs of the molecules. Ideally, the protein molecules have a homogeneous 3D structure but exhibit different orientations in the micrographs. These micrographs are digitized and processed in computers as "single particles". Using two-dimensional alignment and classification techniques, homogenous molecules in the same views are clustered into classes. Their averages enhance the signal of the molecule's 2D shapes. After we assign the particles with the proper relative orientation (Euler angles), we will be able to reconstruct the 2D particle images into a 3D virtual volume.

In single particle 3D reconstruction, an essential step is to correctly assign the proper orientation of each single particle. There are several methods to assign the view for each particle, including the angular reconstitution¹ and random conical tilt (RCT) method². In this protocol, we describe our practice in getting the 3D reconstruction of yeast exosome complex using negative staining EM and RCT. It should be noted that our protocol of electron microscopy and image processing follows the basic principle of RCT but is not the only way to perform the method. We first describe how to embed the protein sample into a layer of Uranyl-Formate with a thickness comparable to the protein size, using a holey carbon grid covered with a layer of continuous thin carbon film. Then the specimen is inserted into a transmission electron microscope to collect untilted (0-degree) and tilted (55-degree) pairs of micrographs that will be used later for processing and obtaining an initial 3D model of the yeast exosome. To this end, we perform RCT and then refine the initial 3D model by using the projection matching refinement method³.

Protocol

1. Principle of the Random Conical Tilt Method

1. The principle of the random conical tilt method requires taking a pair of micrographs of the same region of specimen inside the electron microscope. One picture is taken of the specimen in an untilted position (Figure 1A) and the other picture is taken of the specimen tilted at an angle between 50 to 70 degrees (in our case, we use 55 degrees). (Figure 1B)
2. Using the computer, the digitized micrograph pair is put side by side and images from the same particles are selected. (Figure 1C)
3. In three-dimensional coordinates, the images of untilted particles and their tilted partners are correlat....

Discussion

In this article we present a detailed protocol of sample preparation and three dimensional reconstruction of the exosome complex using negative staining electron microscopy. Using this method, we obtained the 3D reconstruction using random conical tilt method without any prior knowledge of the structure. Random conical tilt method does not necessarily require a homogeneous sample but the following projection matching refinement step would need a homogeneous specimen in order to achieve high resolution.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polyvinyl Formal Resin	Electron Microscopy Sciences	63450-15-7
Uranyl Formate	Electron Microscopy Sciences	22451
Superfrost Microscope Slides	Thermo Fisher Scientific, Inc.	4951F-001
400 mesh grid regular	SPI Supplies	3040C
Carbon coater Auto 306	Edwards Lifesciences
Tecnai-12 Electron Microscope	FEI
Glow Discharger	BAL-TEC		Sputter Coater SCD 005