Cryo-EM single-particle analysis expanding towards increasingly native samples

The explosion of cryo-electron microscopy (cryo-EM) over the last decade has brought with it a range of new approaches for gaining high-resolution structural information on previously inaccessible biological systems. Cryo-EM single-particle analysis (SPA) approaches typically entail overexpression and purification of the target protein. Larger and more complex molecular assemblies often require extensive optimization of the expression, purification and reconstitution procedures. Additionally, prior knowledge of the composition of the structure of interest is required. Approaches employing cryo-focused ion beam (FIB) milling and cryo-electron tomography (cryo-ET) have proven incredibly useful for exploring protein structures within cells while maintaining near-native conditions. Such strategies avoid purification of the target protein or protein complex, yet are often still limited in throughput and achievable resolution. Here, we highlight recent studies demonstrating that the range of samples suitable for SPA is expanding towards increasingly more native samples. We specifically focus on studies investigating complex macromolecular assemblies where tailored sample-preparation strategies made them amenable for SPA, while still keeping them in close-to-native conditions. These examples show that SPA has become a discovery tool for de novo protein identification and complex stoichiometry in more complex and thicker samples.