Advances in Cryo-EM SPA: Tailored Sample Prep for High-Resolution Native Structures

What is it about?

This article reviews recent advances and methodological developments in cryo-EM single-particle analysis (SPA) for structural biology, particularly highlighting its expansion to more complex and near-native biological samples. It outlines the transformative impact of direct electron detectors and improvements in image-analysis software, which have enabled near-atomic resolution structure determination of large protein assemblies via SPA. The article discusses the stringent sample requirements for SPA, such as particle purity, homogeneity, and suitable ice thickness, and contrasts these with alternative strategies like cryo-electron tomography (cryo-ET) and cryo-focused ion beam (cryo-FIB) milling that allow in situ structural studies. It underscores the challenges associated with maintaining native environments, overcoming sample thickness limitations, and achieving high resolution in crowded or heterogeneous cellular contexts. The review also highlights emerging hybrid methods that blur the boundaries between SPA and cryo-ET, such as combining high-dose imaging with tilt-series or applying SPA directly to minimally isolated cellular structures. Notably, the article describes tailored sample-preparation protocols that enable SPA data collection from naturally abundant assemblies with minimal disruption, expanding the applicability of SPA to previously inaccessible targets. Overall, this review emphasizes both the methodological innovations and the increasing versatility of SPA in addressing complex structural biology questions.

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Why is it important?

This review summarises recent advances in cryo-electron microscopy (cryo-EM) single-particle analysis (SPA), with a specific focus on innovative sample preparation techniques that expand the range of biological targets amenable to high-resolution structural investigation. The article discusses how these developments are enhancing the ability to study complex or previously inaccessible molecular assemblies, bridging the gap between structural and cellular biology, and increasingly allowing for structural analysis within near-native environments. Key Takeaways: 1. This review article highlights how the integration of direct electron detectors and advanced image-analysis software has enabled SPA cryo-EM to routinely achieve near-atomic resolution, making it a prominent tool for structural biology, particularly for large proteins, complexes, and membrane-associated assemblies. 2. The article details current challenges in SPA sample preparation, such as the need for protein purification, adequate particle distribution, and optimal ice thickness, and reviews recent strategies—including the use of crude lysates, minimal isolation protocols, and in situ imaging with cryo-electron tomography—that aim to preserve native environments and broaden the applicability of SPA. 3. The review compiles examples where tailored sample-preparation protocols have enabled SPA to be used for de novo identification and high-resolution analysis of complex molecular assemblies directly from cellular sources, demonstrating that the boundaries between SPA and cryo-electron tomography are becoming increasingly blurred as new hybrid and context-preserving approaches emerge.