Cryo-electron microscope observing protein formation.

Unlocking the Secrets of Life: How Cryo-Electron Microscopy is Revolutionizing Structural Biology

Reese Marlowe in Science & Nature September 2025 • 5 min read.

"Discover how cryo-EM is changing our understanding of proteins and their dynamic behaviors, offering new insights into disease and potential treatments."

The 2017 Nobel Prize in Chemistry recognized the pioneers of cryo-electron microscopy (cryo-EM), a method that has reshaped how we visualize the intricate world of proteins. Traditionally, X-ray crystallography and NMR spectroscopy were the dominant techniques for determining protein structures. However, thanks to decades of dedicated effort by numerous scientists, cryo-EM has emerged as a powerful third option [1].

Cryo-EM distinguishes itself by directly imaging protein samples using an electron microscope. This eliminates the need for crystallization or isotopic labeling, allowing researchers to study proteins in a more native-like state. Furthermore, cryo-EM requires only minute amounts of sample and is particularly well-suited for analyzing large protein complexes [1].

Recent advancements have extended cryo-EM's capabilities to dynamic structural analysis, providing insights into how proteins change their conformations in response to various stimuli. Unlike other methods, cryo-EM imposes minimal spatial constraints, making it ideal for studying the dynamic behavior of large protein complexes and capturing structural changes during ligand binding or protein-protein interactions. By analyzing the proportions of different dynamic states, researchers can even glean information about the kinetics of molecular processes.

The Principles of Cryo-Electron Microscopy: How Does It Work?

Cryo-electron microscope observing protein formation.

Biological samples, like proteins, typically rely on water to maintain their shape and function. However, electron microscopes operate under high vacuum conditions (below 10⁻⁵ Pa) to allow electrons to travel freely. Introducing a hydrated biological sample into this environment would cause it to dry out and collapse, losing its native structure and function. Additionally, biological molecules are primarily composed of light elements like carbon, which scatter electrons weakly [2].

This low scattering probability makes them nearly transparent to the electron beam, resulting in low contrast images. Moreover, these light elements are susceptible to radiation damage from the electron beam, leading to structural degradation with even minimal exposure.

Traditional Approaches: Earlier electron microscopy of biological samples relied on techniques like negative staining, where samples were embedded in heavy metal salts like uranyl acetate (Figure 2a). This allowed for visualization based on differences in electron scattering between the heavy metal stain and the sample [3].
Limitations of Negative Staining: While useful, negative staining only provided information about the sample's outline and lacked the ability to resolve internal structural details at high resolution, preventing the determination of atomic coordinates.
Cryo-EM's Solution: Cryo-EM overcomes these limitations by employing a technique called cryo-protection, developed by Jacques Dubochet, one of the Nobel laureates. This involves rapidly freezing the biological sample in a thin film of amorphous ice, effectively trapping it in a near-native state (Figure 2b). This vitrified sample is then loaded into the electron microscope and imaged [4].

To compensate for the inherently low contrast of vitrified samples, cryo-EM utilizes phase contrast. By exploiting the spherical aberration of the electron microscope's objective lens, a slight defocus is introduced, creating a phase shift of π/2 between transmitted and scattered electrons [5]. This enhances the contrast in the image, which can then be further processed to correct for contrast transfer function (CTF) oscillations and recover high-resolution structural information. Unlike negative staining, cryo-EM allows for the acquisition of structural information from the entire sample, marking a significant advancement in protein structure analysis.

Looking Ahead: The Future of Cryo-EM

This review has highlighted the power of single-particle cryo-EM as a leading method for protein structure determination. Its minimal sample requirements and ability to study proteins in near-native conditions pave the way for exciting future applications. Besides single-particle analysis, cryo-electron tomography and subtomogram averaging offer alternative approaches, enabling the study of heterogeneous samples and proteins within cellular contexts, although with slightly reduced resolution. As cryo-EM technology continues to advance, expect even more exciting developments and broader adoption across various scientific disciplines.

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Everything You Need To Know

What is cryo-electron microscopy and how does it work?

Cryo-electron microscopy (cryo-EM) is a technique that allows scientists to visualize the structure of proteins and other biological molecules. Unlike traditional methods, it images samples directly using an electron microscope without the need for crystallization or isotopic labeling. This is significant because it allows proteins to be studied in a more native-like state, providing a more accurate understanding of their structure and function. This is especially important for studying large protein complexes and dynamic behaviors, which are challenging to analyze with other methods. The implications are far-reaching, including advancements in drug discovery and understanding disease mechanisms.

Why is cryo-EM considered an important advancement in studying proteins?

Cryo-EM is important because it overcomes the limitations of traditional techniques like X-ray crystallography and NMR spectroscopy, which are used to determine protein structures. X-ray crystallography requires that proteins be crystallized, which can be difficult or impossible for many proteins, especially large complexes. NMR spectroscopy has limitations regarding the size of the protein that can be studied. Cryo-EM eliminates the need for crystallization or isotopic labeling and requires minute amounts of sample, making it suitable for a broader range of protein studies. This enables scientists to study proteins in near-native conditions, capturing structural changes during processes like ligand binding or protein-protein interactions, offering valuable insights into molecular processes.

What is cryo-protection and why is it used in cryo-EM?

Cryo-protection is a key aspect of cryo-electron microscopy (cryo-EM). It involves rapidly freezing a biological sample in a thin film of amorphous ice, preserving it in a near-native state. This is crucial because electron microscopes operate under high vacuum conditions, which would cause hydrated biological samples to dry out and collapse, losing their structure. Cryo-protection, developed by Jacques Dubochet, one of the Nobel laureates, allows scientists to image proteins without damaging them. The implications are that it enables the study of biological structures at high resolution, offering a more accurate view of the molecules and their functions.

What is phase contrast and why is it important in cryo-EM?

Phase contrast is a technique used in cryo-EM to enhance the image contrast of vitrified samples. Biological samples have low contrast because they are composed of light elements that weakly scatter electrons. By exploiting the spherical aberration of the electron microscope's objective lens, a slight defocus is introduced, creating a phase shift of π/2 between transmitted and scattered electrons. This enhances the contrast in the image. This is significant because it allows for the acquisition of structural information from the entire sample, improving the resolution and quality of the images. The implications are that phase contrast enhances the visualization of the sample's internal structures, and allows scientists to obtain detailed structural information that would be difficult or impossible to achieve otherwise.

What are some different approaches using cryo-EM?

Single-particle cryo-EM is a key approach in this field. It involves averaging images of individual protein particles to generate a high-resolution structure. This method has minimal sample requirements and allows proteins to be studied in near-native conditions. Other approaches, such as cryo-electron tomography and subtomogram averaging, provide alternative methods for studying heterogeneous samples and proteins within cellular contexts, albeit with slightly reduced resolution. The implications are a deeper understanding of proteins and their functions, leading to advancements in fields like drug discovery and disease research. As technology advances, cryo-EM will be adopted across many different scientific disciplines.