Combining high throughput and high quality for cryo-electron microscopy data collection

Abstract

Cryo-electron microscopy (cryo-EM) can be used to elucidate the 3D structure of macromolecular complexes. Driven by technological breakthroughs in electron-microscope and electron-detector development, coupled with improved image-processing procedures, it is now possible to reach high resolution both in single-particle analysis and in cryo-electron tomography and subtomogram-averaging approaches. As a consequence, the way in which cryo-EM data are collected has changed and new challenges have arisen in terms of microscope alignment, aberration correction and imaging parameters. This review describes how high-end data collection is performed at the EMBL Heidelberg cryo-EM platform, presenting recent microscope implementations that allow an increase in throughput while maintaining aberration-free imaging and the optimization of acquisition parameters to collect high-resolution data.

Keywords: coma-free imaging; cryo-electron microscopy; fringe-free imaging; high-end data collection.

Figure 1

Fringe-free illumination. (a, b) Image of the beam without (a) and with (b) fringe-free illumination. The beam diameter is 460 nm and the images were recorded at spot 9 over a 20 s exposure with a pixel size of 1.34 Å. (c, d) Acquisition scheme without (c) and with (d) fringe-free illumination. The sample is embedded in a thin layer of ice over a holey carbon film with 2 µm diameter holes. The blue squares represent the imaged area in the context of a 1.04 Å pixel. Without fringe-free illumination (c) the beam size, depicted by an orange circle, needs to be ∼1 µm in diameter in order to avoid seeing fringes within the imaged area, limiting the number of acquisitions to five within the hole. In the case of fringe-free illumination (d), a beam size of 600 nm is enough to cover the camera, allowing up to ten acquisitions.