Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6

Abstract

Biological specimens suffer radiation damage when imaged in an electron microscope, ultimately limiting the attainable resolution. At a given resolution, an optimal exposure can be defined that maximizes the signal-to-noise ratio in the image. Using a 2.6 Å resolution single particle cryo-EM reconstruction of rotavirus VP6, determined from movies recorded with a total exposure of 100 electrons/Å ², we obtained accurate measurements of optimal exposure values over a wide range of resolutions. At low and intermediate resolutions, our measured values are considerably higher than obtained previously for crystalline specimens, indicating that both images and movies should be collected with higher exposures than are generally used. We demonstrate a method of using our optimal exposure values to filter movie frames, yielding images with improved contrast that lead to higher resolution reconstructions. This ‘high-exposure’ technique should benefit cryo-EM work on all types of samples, especially those of relatively low-molecular mass.

DOI: http://dx.doi.org/10.7554/eLife.06980.001

eLife digest

Microscopes allow us to visualize objects that are invisible to the naked eye. One type of microscope—called the electron microscope—produces images using beams of particles known as electrons, which enables them to produce more detailed images than microscopes that use light.

There are several ways to prepare samples for electron microscopy. For example, in ‘electron cryo-microscopy’—or cryo-EM for short—a sample is rapidly frozen to preserve its features before it is examined under the microscope. This technique generates images that can be analyzed by computers to produce three-dimensional models of individual viruses, proteins, and other tiny objects. Unfortunately, the samples need to be exposed to high-energy beams of electrons that will damage the sample while the images are gathered, which results in sample movement and blurry images that lack the finer details.

The contrast between the sample and its background is one of the factors that determine the final quality of an image. The higher the contrast, the greater the level of structural information that can be obtained, but this requires the use of longer exposures to the electron beam. To overcome this issue, researchers found that instead of recording a single image, it is possible to record movies in which the movement of the sample under the electron beam can be tracked. After the movies are gathered, the movie frames are aligned using computer software to reduce the blurring caused by the sample moving and can then be used to make three-dimensional models.

Grant and Grigorieff improved this method further by studying how quickly a large virus-like particle called ‘ rotavirus double-layered particle’ is damaged under the electron beam. These experiments identified an optimum range of exposure to electrons that provides the highest image contrast at any given level of detail. These findings were used to design an exposure filter that can be applied to the movie frames, allowing Grant and Grigorieff to visualize features of the virus that had not previously been observed by cryo-EM.

This method was also used to study an assembly of proteins known as the proteasome, which is responsible for destroying old proteins. Grant and Grigorieff's findings should be useful for cryo-EM studies on many kinds of samples.

DOI: http://dx.doi.org/10.7554/eLife.06980.002