How low can we go? Structure determination of small biological complexes using single-particle cryo-EM

Abstract

For decades, high-resolution structural studies of biological macromolecules with masses of <200kDa by cryo-EM single-particle analysis were considered infeasible. It was not until several years after the advent of direct detectors that the overlooked potential of cryo-EM for studying small complexes was first realized. Subsequent advances in sample preparation, imaging, and data processing algorithms have improved our ability to visualize small biological targets. In the past two years alone, nearly two hundred high-resolution structures have been determined of small (<200kDa) macromolecules, the smallest being approximately 39kDa in molecular weight. Here we summarize some salient lessons and strategies for cryo-EM studies of small biological complexes, and also consider future prospects for routine structure determination.

Figure 1

High-resolution sub-200 kDa structures determined by cryo-EM single-particle analysis. Top: Electron Microscopy Data Bank (EMDB) entries of complexes amassing below 200 kDa and resolved to better than 4 Å resolution, plotted by molecular weight and resolution. Data points are up to date to the point of this publication. Bottom: Cryo-EM density is shown for selected entries (outlined in black in the plot above) along with the corresponding EMDB identifier, molecule name, molecular mass, and reported resolution. Further details for each structure are listed in Table S1.

Figure 2

Cryo-EM reconstructions of 64 kDa hemoglobin and 52 kDa streptavidin imaged with and without a Volta Phase Plate (VPP). Left: Hemoglobin determined to 2.8 Å resolution using conventional defocus-based imaging (top) and to 3.2 Å using a VPP (bottom). Right: Streptavidin determined to 2.6 Å resolution using conventional defocus-based imaging (top) and to 3.2 Å using a VPP (bottom). The full map and density features (with the corresponding atomic model docked in) are shown for each structure.

Figure 3

Flowchart for cryo-EM SPA analysis of small biological complexes. Important considerations for structure determination of small targets at each stage of frozen specimen preparation, data acquisition, and image processing are highlighted. Potential pitfalls and possibilities for overcoming them (as demonstrated by published work) are also indicated.