Using Cryo-EM to Determine the Structure of Macromolecular Complexes
Cryo-electron microscopy (cryo-EM) transforms the way we visualize biological molecules. The ability to discern biological structures preserved in their natural state at near- or sometimes true atomic resolution gives scientists invaluable insights into the function of large and also dynamic macromolecules. Unlike X-ray crystallography, which requires crystallization, cryo-EM can be used on flexible molecular assemblies, allowing researchers to study complex biological systems that would otherwise remain out of reach. Success in cryo-EM largely depends on the preservation of biological molecules in a state as close to their native environment as possible. This advantage makes cryo-EM indispensable in revealing molecular interactions that are crucial for understanding biological processes.
Single Particle Analysis: Reconstructing 3D Structures
Single particle analysis (SPA) is a cryo-EM method used to determine the structure of vitrified biomolecules. This technique involves collecting multiple 2D projections of ideally randomly oriented particles. Advanced image processing software aligns these images and computationally reconstructs a detailed 3D structure. An advantage of SPA lies in its ability to capture various conformations of the same molecule, making it particularly useful for studying dynamic complexes. Moreover, sophisticated algorithms help handle challenges like sample heterogeneity and molecular flexibility, allowing scientists to obtain high-resolution structures of otherwise challenging biomolecules.
Advantages Over Traditional Methods
Cryo-EM offers several distinct advantages over other structure determination techniques such as X-ray crystallography and nuclear magnetic resonance (NMR). As mentioned above, cryo-EM does not require crystallization. Additionally, cryo-EM provides a more realistic depiction of biological molecules in their near-native environment. It can also capture macromolecules in multiple conformational states, offering insights into their functional dynamics. We'll discuss this more shortly. Also, cryo-EM can yield detailed structures from tiny amounts of material, often no more than a few micrograms.
Compared to NMR, cryo-EM can handle much larger complexes, as NMR struggles with molecules over 100 kDa due to signal overlap and complexity. These advantages have made cryo-EM an essential tool for structural biologists aiming to study complex biological systems, such as membrane proteins and other large assemblies.
How Does Cryo-EM Handle the Heterogeneity of Macromolecular Complexes?
As mentioned, cryo-EM's ability to handle the inherent heterogeneity of macromolecular complexes is a significant advantage. Biological molecules often exist in multiple conformational states, which can result in structural flexibility and variation. This heterogeneity presents challenges for researchers aiming to achieve high-resolution structures, but cryo-EM offers several computational and experimental strategies to manage this complexity.
- Computational Approaches: Cryo-EM employs advanced computational techniques to manage heterogeneity. 3D classification algorithms sort particles into groups based on structural similarities, allowing for the reconstruction of multiple structures from within the same dataset. This technique is routinely used to isolate distinct conformational or compositional states. Masking techniques also allow researchers to focus within specific regions of a structure and improve the resolution of those regions while ignoring the signal from the more flexible parts. Finally, machine learning algorithms are increasingly being used to improve classification of heterogeneous data.
- Experimental Approaches: Experimental methods such as biochemical optimization and ligand binding can also help reduce heterogeneity by stabilizing specific functional states. By refining sample preparation techniques, researchers can create more homogeneous conditions, reducing the impact of structural variability on the final reconstruction.
Together, these strategies enable cryo-EM to handle complex, flexible biological systems, offering a comprehensive view of macromolecular dynamics and the biological processes they regulate.
Challenges and Future Directions
Despite its many advantages, cryo-EM is not without its challenges. Sample preparation remains a critical step, with ongoing research focused on optimizing EM grid preparation and vitrification techniques to improve sample quality. Additionally, while cryo-EM has achieved remarkable resolution with large complexes, it faces limitations when applied to smaller proteins (less than 100 kDa). Addressing these challenges may involve further advances in detector sensitivity, image processing algorithms, and microscope hardware.
Nevertheless, the future of cryo-EM is promising as researchers continue to push the boundaries of what can be achieved with this technique. As technology improves, cryo-EM is expected to extend its reach into smaller proteins and more complex biological systems, further cementing its role as a
cornerstone of structural biology and drug discovery.References and further reading
- Azinas S, Carroni M. Cryo-EM uniqueness in structure determination of macromolecular complexes: A selected structural anthology. Curr Opin Struct Biol. 2023 Aug;81:102621. doi: 10.1016/j.sbi.2023.102621. Epub 2023 Jun 12. PMID: 37315343.
- Holger Stark, Ashwin Chari, Sample preparation of biological macromolecular assemblies for the determination of high-resolution structures by cryo-electron microscopy, Microscopy, Volume 65, Issue 1, February 2016, Pages 23–34, https://doi.org/10.1093/jmicro/dfv367
- Carroni M, Saibil HR. Cryo electron microscopy to determine the structure of macromolecular complexes. Methods. 2016 Feb 15;95:78-85. doi: 10.1016/j.ymeth.2015.11.023. Epub 2015 Nov 27. PMID: 26638773; PMCID: PMC5405050.
- Jonić S. Cryo-electron Microscopy Analysis of Structurally Heterogeneous Macromolecular Complexes. Comput Struct Biotechnol J. 2016 Oct 14;14:385-390. doi: 10.1016/j.csbj.2016.10.002. PMID: 27800126; PMCID: PMC5072154.