Present and Emerging Methodologies in Cryo-EM Single-Particle Analysis

Abstract

Over the past decade, technical and methodological improvements in cryogenic electron microscopy (cryo-EM) single-particle analysis have enabled routine high-resolution structural analyses of biological macromolecules, resulting in a flood of new molecular insights into protracted biological questions. However, despite the tremendous progress and success of the field in recent years, opportunities for improvement remain in various aspects of the cryo-EM single-particle analysis workflow (e.g., sample preparation, image acquisition and processing, and structure validation). Here, we review recent advances that have contributed to the principal methods in cryo-EM and identify persisting challenges and bottlenecks that will require further methodological and hardware development.

Figure 1

Resolution and map deposition trends in single-particle cryo-EM. Shown are the annual (blue dotted curve) and cumulative (orange curve) map depositions into the Electron Microscopy Data Bank (EMDB). The median resolution of deposited maps per year is also shown (gray curve); future cryo-EM methodological and hardware developments, such as those discussed in this review, will likely improve the median resolution to 4 Å or better over the next decade. To see this figure in color, go online.

Figure 2

Cryo-EM structures of biological macromolecules enabled by technical and methodological advances in SPA. Top: shown is mouse apoferritin determined to ∼1.2-Å resolution using a TEM equipped with a cold FEG and next-generation energy filter and direct detector. Middle: shown is the ∼2.6-Å resolution reconstruction of the 52-kDa streptavidin imaged over graphene monolayer support grids. Bottom: shown is the ∼4.2-Å resolution reconstruction of the substrate-engaged yeast 26S proteasome in the "4D" motor state. Focused classification and refinement strategies (colored by focusing area) were used to identify the distinct motor conformations and to generate composite reconstructions to facilitate model building for each state. The inset on the right shows density features of the substrate polypeptide encircled by a spiral-staircase arrangement of pore loop tyrosine residues within the central pore of the AAA+ motor. The EMDB accession code and full map (left) and density features with the corresponding atomic model docked in (right) are shown for each structure. To see this figure in color, go online.

Figure 3

Current bottlenecks in the cryo-EM SPA workflow. Aspects of the workflow that impose significant limitations to efficiency, data quality, and/or reproducibility are indicated, along with potential future opportunities for reducing these bottlenecks. To see this figure in color, go online.