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. The article discusses the 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 proposes that the boundaries between SPA and cryo-ET are currently blurring. 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 the increasing versatility of SPA in addressing complex structural biology questions and illustrates this with timely examples pushing the boundaries of SPA.

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Photo by CDC on Unsplash

Photo by CDC on Unsplash

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. 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. While the integration of direct electron detectors and advanced image-analysis software have enabled SPA cryo-EM to routinely achieve near-atomic resolution, several challenges in SPA sample preparation, such as the need for protein purification, adequate particle distribution, and optimal ice thickness, remain. 2. The review highlights 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. These examples demonstrate that recent improvements in SPA are closing the gap to obtaining high-resolution structural information in situ.