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How does cryo-EM advance protein structure determination? ​

Abstract ​

Cryo-electron microscopy (cryo-EM) has emerged as a transformative technology in structural biology, enabling researchers to elucidate the architecture of proteins and protein complexes at unprecedented resolutions. This advancement is particularly significant given the limitations associated with traditional techniques such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, which often struggle with complex biological samples. The ability of cryo-EM to visualize biological macromolecules in their near-native states has opened new avenues for understanding fundamental biological processes, particularly in the context of drug design and disease mechanisms. The significance of cryo-EM lies not only in its capacity to produce high-resolution structures but also in its potential to address longstanding challenges in structural biology. The technique facilitates the study of large and dynamic macromolecular assemblies that are often difficult to crystallize or purify, thus providing insights into their functional states. Recent advancements in cryo-EM technology, driven by improvements in instrumentation and sample preparation techniques, have led to resolutions better than 4 Å, with some studies achieving atomic-resolution structures. This review synthesizes current knowledge and emerging trends in cryo-EM, emphasizing its pivotal role in modern biomedical research and its potential for future advancements in structural biology. The review is organized to provide an overview of cryo-EM technology, recent advances, key case studies, a comparison with traditional methods, and future perspectives, underscoring the technique's transformative impact on protein structure determination and drug discovery.

Outline ​

This report will discuss the following questions.

  • 1 Introduction
  • 2 Overview of Cryo-EM Technology
    • 2.1 Historical Development of Cryo-EM
    • 2.2 Basic Principles of Cryo-EM
  • 3 Advances in Cryo-EM Techniques
    • 3.1 Improvements in Detector Technology
    • 3.2 Innovations in Sample Preparation
    • 3.3 Enhanced Image Processing Algorithms
  • 4 Case Studies: Cryo-EM in Action
    • 4.1 Structural Determination of Membrane Proteins
    • 4.2 Insights into Protein Complexes
    • 4.3 Applications in Drug Discovery
  • 5 Comparison with Traditional Methods
    • 5.1 Cryo-EM vs. X-ray Crystallography
    • 5.2 Cryo-EM vs. NMR Spectroscopy
  • 6 Future Perspectives in Cryo-EM
    • 6.1 Potential Developments in Technology
    • 6.2 Expanding Applications in Structural Biology
  • 7 Summary

1 Introduction ​

Cryo-electron microscopy (cryo-EM) has emerged as a transformative technology in structural biology, enabling researchers to elucidate the architecture of proteins and protein complexes at unprecedented resolutions. This advancement is particularly significant given the limitations associated with traditional techniques such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, which often struggle with complex biological samples. The ability of cryo-EM to visualize biological macromolecules in their near-native states has opened new avenues for understanding fundamental biological processes, particularly in the context of drug design and disease mechanisms [1][2].

The significance of cryo-EM lies not only in its capacity to produce high-resolution structures but also in its potential to address longstanding challenges in structural biology. The technique facilitates the study of large and dynamic macromolecular assemblies that are often difficult to crystallize or purify, thus providing insights into their functional states [3][4]. As the demand for structural insights into proteins increases, especially in relation to therapeutic development, cryo-EM has established itself as a critical tool in the biomedical research landscape [5].

The current state of cryo-EM technology reflects a rapid evolution over the past decade, driven by significant advancements in instrumentation, including the development of direct electron detectors and sophisticated image processing algorithms [4][6]. These improvements have resulted in the ability to achieve resolutions better than 4 Å, with some recent studies reporting atomic-resolution structures [2]. Moreover, innovations in sample preparation techniques have enhanced the visualization of challenging targets, such as membrane proteins and large protein complexes [3][5].

This review is organized to provide a comprehensive overview of the advancements in cryo-EM technology and its implications for protein structure determination. The following sections will cover:

  1. An overview of cryo-EM technology, including its historical development and basic principles.
  2. Recent advances in cryo-EM techniques, highlighting improvements in detector technology, innovations in sample preparation, and enhanced image processing algorithms.
  3. Key case studies illustrating the application of cryo-EM in structural determination, particularly focusing on membrane proteins, protein complexes, and its role in drug discovery.
  4. A comparison of cryo-EM with traditional structural biology methods, specifically X-ray crystallography and NMR spectroscopy, to delineate their respective strengths and weaknesses.
  5. Future perspectives on cryo-EM, including potential technological developments and expanding applications in structural biology.

By synthesizing current knowledge and emerging trends in cryo-EM, this review aims to underscore the technique's pivotal role in modern biomedical research and its potential for future advancements in the field of structural biology [7][8].

2 Overview of Cryo-EM Technology ​

2.1 Historical Development of Cryo-EM ​

Cryo-electron microscopy (cryo-EM) has significantly advanced the field of protein structure determination, evolving into a powerful tool for elucidating the three-dimensional structures of biological macromolecules. The historical development of cryo-EM reflects a trajectory marked by technological innovations and methodological enhancements that have collectively transformed its capabilities.

Initially, cryo-EM emerged as a viable alternative to traditional techniques such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, which often faced limitations in studying larger and more complex biomolecules. Cryo-EM enables the visualization of proteins in their native, hydrated states, allowing for the analysis of structures that are difficult to crystallize. This advantage is particularly crucial for membrane proteins and large macromolecular complexes, which represent a significant portion of druggable targets in pharmacology [5].

The advent of direct electron detectors has been a pivotal advancement in cryo-EM technology. These detectors enhance the quality of the captured images by improving contrast and reducing noise, thereby facilitating the determination of structures at near-atomic resolutions [2]. Furthermore, the development of sophisticated image processing algorithms has allowed for the effective handling of data, enabling the reconstruction of high-resolution density maps from single-particle analyses [6].

Historically, the resolution achievable with cryo-EM has steadily improved, with notable milestones including the determination of structures at resolutions better than 4 Å, which enables atomic model building [2]. Recent reports have demonstrated structures resolved to 1.25 Å, allowing for the visualization of individual atoms and the identification of chemical modifications [2]. This leap in resolution has profound implications for understanding protein function, interactions, and the mechanisms of drug binding [5].

Cryo-EM's integration with other structural biology techniques, such as X-ray crystallography and NMR, has further enhanced its utility. By combining data from cryo-EM with high-resolution models from crystallography, researchers can achieve more comprehensive insights into complex molecular assemblies [9]. This complementary approach facilitates the analysis of dynamic systems and heterogeneous samples, which are often challenging to study with a single technique [7].

The ongoing improvements in cryo-EM methodology continue to push the boundaries of what is possible in structural biology. Future developments are expected to further enhance resolution, reduce costs, and simplify the technical barriers to entry, thereby broadening the accessibility of this powerful technique [1]. As cryo-EM technology matures, its role in drug discovery and the understanding of biological processes is poised to expand, offering new avenues for therapeutic development and the elucidation of fundamental biological mechanisms [10].

2.2 Basic Principles of Cryo-EM ​

Cryo-electron microscopy (cryo-EM) has significantly advanced the field of protein structure determination by providing a powerful technique capable of elucidating the three-dimensional structures of biological macromolecules at near-atomic resolution. The evolution of cryo-EM technology has enabled researchers to overcome many of the limitations associated with traditional structural biology methods such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.

The basic principles of cryo-EM involve the rapid freezing of biological samples to preserve their native state, followed by imaging using electron microscopy. This technique allows for the direct observation of proteins in a near-physiological environment without the need for crystallization, which can be a major bottleneck in structural studies. Cryo-EM is particularly adept at analyzing large and complex protein assemblies, which are often difficult to crystallize. The ability to visualize proteins in their functional forms has made cryo-EM a preferred method for studying dynamic biological processes.

Recent advancements in cryo-EM technology include improvements in microscope design, detector technology, and image processing software, which have collectively enhanced the resolution and quality of the data obtained. For instance, the development of direct electron detection cameras has led to significant gains in image quality, allowing for the determination of protein structures at resolutions of approximately 2 Å or even higher in some cases [6]. These improvements facilitate the visualization of protein structures at unprecedented detail, enabling researchers to observe individual atoms and their interactions within protein complexes [2].

Cryo-EM has also integrated with other structural determination techniques, such as X-ray crystallography and NMR spectroscopy, to provide complementary data that enhance the overall understanding of protein structures. For example, cryo-EM density maps can be used alongside crystallographic data to achieve atomic-resolution models of complex molecular assemblies [9]. This integrative approach not only increases the accuracy of structural models but also expands the range of biological questions that can be addressed.

Moreover, the methodological advancements in cryo-EM have improved sample preparation techniques, which are crucial for studying membrane proteins and other challenging targets [3]. The ability to mimic the lipid environment of membrane proteins has led to more accurate structural determinations, thus broadening the scope of proteins that can be studied using cryo-EM [5].

In summary, cryo-EM has revolutionized protein structure determination by providing a versatile and powerful tool that allows for high-resolution analysis of complex biological macromolecules. Its capacity to visualize proteins in their native states, coupled with continuous technological advancements, positions cryo-EM as a cornerstone technique in modern structural biology, facilitating insights into protein function, dynamics, and interactions critical for drug design and therapeutic development [4][10].

3 Advances in Cryo-EM Techniques ​

3.1 Improvements in Detector Technology ​

Cryo-electron microscopy (cryo-EM) has significantly advanced protein structure determination through a series of technological improvements, particularly in detector technology. Recent developments in direct electron detection have transformed cryo-EM into a powerful method for elucidating the structures of biological macromolecules at near-atomic resolutions. This advancement addresses several historical limitations associated with cryo-EM, such as resolution and image quality.

The introduction of direct electron detectors has led to enhanced sensitivity and resolution in cryo-EM imaging. These detectors can capture high-quality images of biological specimens with reduced electron dose, which is crucial for preserving the structural integrity of sensitive macromolecules during imaging. For instance, cryo-EM has reached resolutions of better than 4 Å, allowing for atomic model building and visualization of individual atoms within proteins, as reported by Yip et al. (2020) who achieved a 1.25 Å resolution structure of apoferritin, showcasing the unprecedented detail now attainable in cryo-EM studies[2].

Moreover, the integration of advanced image processing software has complemented the improvements in detector technology. This software enhances the quality of cryo-EM density maps by correcting for various artifacts and noise, enabling researchers to derive more accurate structural information from the data collected. As noted by Danev et al. (2019), the combination of these advancements has positioned cryo-EM as a leading technique for determining the structures of complex and dynamic protein assemblies[1].

In addition to the technological enhancements in detectors, cryo-EM's capability to visualize proteins in their native state has opened new avenues for structural biology. It allows for the study of proteins that are difficult to crystallize, thus expanding the range of macromolecules that can be analyzed. This is particularly beneficial for membrane proteins and large macromolecular complexes, which are often key targets in drug discovery[11].

Overall, the advancements in cryo-EM, particularly through improvements in detector technology, have revolutionized protein structure determination by enabling high-resolution imaging of a wider variety of biological structures, thereby facilitating significant progress in understanding protein function and dynamics. The continual refinement of cryo-EM methodologies promises to further enhance its utility in structural biology and drug discovery[12].

3.2 Innovations in Sample Preparation ​

Cryo-electron microscopy (cryo-EM) has made significant advancements in protein structure determination, particularly through innovations in sample preparation techniques. These improvements have addressed long-standing challenges associated with visualizing proteins and their complexes at high resolutions, thereby expanding the capabilities of cryo-EM as a structural biology tool.

One of the critical developments in sample preparation is the enhancement of specimen preparation methods, which has been crucial for obtaining high-resolution images. For instance, the integration of direct electron detectors and improved sample handling protocols has facilitated the acquisition of clearer images of biological samples. These advancements allow researchers to analyze proteins that were previously difficult to study due to issues related to crystallization or low protein yield [3].

Additionally, specific techniques aimed at mimicking the lipid environment of membrane proteins have been developed. Traditional methods often involved solubilizing membrane proteins with detergents, which can diminish contrast and complicate the resolution of cryo-EM studies. Recent innovations focus on creating conditions that better replicate the native lipid bilayer environment, significantly improving the quality of structural data obtained from membrane proteins [3].

The use of cryo-electron tomography (cryo-ET) is another innovation that complements single-particle analysis. Cryo-ET allows for the visualization of macromolecular complexes in their native cellular environment, providing unprecedented detail that is essential for understanding the dynamics and interactions of these complexes [6]. This method leverages tilt series of images to obtain 3D reconstructions, enhancing the ability to resolve structures that exhibit conformational variability [13].

Furthermore, methodological advancements such as constrained single-particle tomography have emerged, which utilize geometric constraints to improve the accuracy of molecular orientation assignments necessary for 3D reconstruction. This technique helps reduce model bias and artifacts, enabling the determination of protein structures at resolutions that were previously unattainable [13].

Overall, the innovations in sample preparation and associated methodologies in cryo-EM are pivotal in advancing the resolution and accuracy of protein structure determination. These improvements not only enhance the understanding of protein dynamics and interactions but also facilitate the exploration of complex biological systems that were once considered intractable [1][4]. As cryo-EM continues to evolve, it is expected to further impact structural biology and drug discovery significantly, enabling insights into the structural basis of protein function and interaction at unprecedented resolutions [5][10].

3.3 Enhanced Image Processing Algorithms ​

Cryo-electron microscopy (cryo-EM) has significantly advanced the field of protein structure determination through several technological improvements, particularly in the realm of image processing algorithms. The integration of advanced image processing techniques has enhanced the capability of cryo-EM to provide high-resolution structures of biological macromolecules, which was previously challenging due to limitations in resolution and data analysis.

One notable advancement in cryo-EM is the development of direct electron detectors and sophisticated image processing software. These innovations have enabled the determination of structures at near-atomic resolutions, allowing researchers to overcome the challenges associated with traditional methods like X-ray crystallography, which often struggle with complex or flexible protein structures. For instance, single-particle analysis (SPA) has become a prominent method in cryo-EM, enabling the resolution of protein structures with sizes smaller than 100 kDa at resolutions of approximately 2 Å [6].

Furthermore, the introduction of artificial intelligence (AI) in image processing has transformed particle picking—a crucial step in the reconstruction of protein structures from cryo-EM micrographs. Traditional methods of particle picking were labor-intensive and time-consuming, but recent AI-driven approaches have significantly automated this process, improving both precision and efficiency. For example, the development of CryoTransformer, a model based on transformers and image processing techniques, has demonstrated superior performance in accurately picking protein particles, thereby enhancing the quality of reconstructed 3D density maps [14].

The combination of improved hardware, such as high-performance electron detectors, with cutting-edge software for data analysis has also led to a substantial increase in the number of structures solved annually. This synergy has made cryo-EM a competitive alternative to other structural biology techniques, facilitating the visualization of dynamic and heterogeneous macromolecular complexes in their native environments [4].

In summary, advancements in cryo-EM, particularly in enhanced image processing algorithms and AI integration, have revolutionized protein structure determination by improving resolution, automating particle picking, and enabling the analysis of complex biological systems that were previously difficult to study. These developments not only expand the capabilities of cryo-EM but also open new avenues for structural biology and drug design, ultimately contributing to a deeper understanding of protein function and interaction mechanisms [5][10].

4 Case Studies: Cryo-EM in Action ​

4.1 Structural Determination of Membrane Proteins ​

Cryo-electron microscopy (cryo-EM) has significantly advanced the field of protein structure determination, particularly for membrane proteins, which are notoriously difficult to study due to their inherent instability and complex environments. The evolution of cryo-EM technology has led to the capability of achieving near-atomic resolution structures without the need for crystallization, thereby opening new avenues for structural biology and drug discovery.

One of the primary advancements in cryo-EM is the development of better instrumentation, including direct electron detectors and improved methods for specimen preparation. These advancements have enabled researchers to solve structures of proteins with sizes smaller than 100 kDa and achieve resolutions of approximately 2 Å in certain cases [6]. The application of single-particle cryo-EM methods has allowed for the visualization of macromolecular complexes in their native environments, overcoming many of the challenges faced in structural and cell biology, such as analyzing highly dynamic soluble and membrane-embedded protein complexes [6].

For membrane proteins specifically, cryo-EM has addressed significant challenges related to their structural determination. Traditionally, these proteins were solubilized and stabilized using various detergents, which unfortunately diminished the contrast of membrane proteins in cryo-EM studies and hindered the acquisition of high-resolution structures [3]. Recent innovations have focused on mimicking the lipid environment of membrane proteins, enhancing their stability and improving resolution [3].

Furthermore, advancements in sample preparation techniques have played a crucial role in the successful application of cryo-EM to membrane proteins. Techniques have evolved to include new solubilization strategies that better stabilize protein complexes and the development of specialized grid supports that minimize molecular motion during electron beam exposure [15]. These improvements have been essential in facilitating the structural determination of membrane proteins, allowing for detailed analysis of their dynamics, interactions with binding partners, and responses to electrochemical gradients [16].

Case studies illustrate the transformative impact of cryo-EM on understanding membrane proteins. For instance, the structural determination of G protein-coupled receptors (GPCRs) has benefited immensely from cryo-EM, providing insights into their molecular interactions with drugs and the mechanisms by which they operate [17]. The structures obtained through cryo-EM have revealed the conformational states of these receptors, elucidating their roles in cellular signaling and drug action [17].

Moreover, cryo-EM has been instrumental in drug discovery, particularly in the rational design of therapeutics targeting membrane proteins [5]. The ability to visualize the structures of integral membrane proteins, which constitute a significant portion of druggable targets, has provided crucial insights into their functions and interactions, thereby facilitating the development of novel therapeutic agents [5].

In conclusion, cryo-EM has revolutionized the structural determination of membrane proteins through advancements in technology and methodology. Its ability to capture high-resolution structures in a near-native state has provided invaluable insights into the function and dynamics of these crucial biomolecules, paving the way for significant advancements in both basic and translational biomedicine.

4.2 Insights into Protein Complexes ​

Cryo-electron microscopy (cryo-EM) has significantly advanced the field of protein structure determination, particularly in the analysis of complex macromolecular assemblies. The evolution of cryo-EM technology, including enhancements in instrumentation, sample preparation, and image processing, has enabled researchers to obtain high-resolution structures of proteins and their complexes that were previously difficult to resolve using traditional methods such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.

One notable advancement in cryo-EM is the development of direct electron detectors and improved image processing algorithms, which have led to the successful determination of protein structures at resolutions as high as 1.25 Å. This level of detail allows for the visualization of individual atoms within proteins, which is crucial for understanding the mechanisms of protein function and interactions with potential drug molecules [2]. For instance, the structure of apoferritin was resolved at 1.25 Å, revealing unprecedented structural details that are critical for applications in structure-based drug design [2].

Cryo-EM's ability to analyze proteins in near-native states is particularly advantageous for studying large and flexible macromolecular complexes. The technique allows for the visualization of protein assemblies in their functional forms without the need for crystallization, which is often a significant bottleneck in structural biology [18]. This is especially important for integral membrane proteins and dynamic complexes, which can be challenging to crystallize [5].

The application of cryo-EM has also proven transformative in the study of complex protein assemblies. For example, single-particle cryo-EM has enabled the visualization of G protein-coupled receptors (GPCRs) and other critical drug targets, providing insights into their structure and dynamics that are essential for drug development [5]. Additionally, the combination of cryo-EM with other structural techniques, such as NMR, has been proposed to enhance the understanding of protein dynamics and conformational changes, thereby providing a more comprehensive view of protein behavior in biological systems [7].

Recent studies have highlighted the utility of cryo-EM in elucidating the structures of heterogeneous macromolecular complexes, capturing multiple conformational states and intermediates that are critical for understanding enzymatic mechanisms and dynamic processes [19]. This capability is particularly valuable in the context of complex biological questions, where traditional structural methods may fall short due to limitations in resolving flexibility and heterogeneity [20].

Overall, cryo-EM represents a powerful tool in structural biology, facilitating the detailed examination of protein complexes in their functional states and advancing our understanding of their roles in biological processes. The ongoing improvements in cryo-EM technology promise to further enhance its application in protein structure determination, paving the way for new discoveries in the field of biomedicine [4].

4.3 Applications in Drug Discovery ​

Cryo-electron microscopy (cryo-EM) has emerged as a transformative technique in the field of structural biology, significantly advancing the determination of protein structures and playing a crucial role in drug discovery. The methodology enables researchers to visualize biological macromolecules at near-atomic resolution, thereby providing detailed insights into their structures and functions.

One of the key advancements in cryo-EM is its ability to analyze large macromolecular complexes, which were previously challenging to study using traditional techniques such as X-ray crystallography. Recent technological developments, including the use of direct electron detectors and advanced image processing algorithms, have enhanced the resolution and quality of cryo-EM images. For instance, structures of complex proteins, including membrane proteins and large assemblies, can now be resolved at resolutions better than 4 Å, with some studies achieving atomic resolution of 1.25 Å for proteins like apoferritin [2]. This level of detail allows for the visualization of individual atoms and even hydrogen atoms within the protein structures, which is critical for understanding the mechanisms of protein-catalyzed reactions and drug interactions [2].

In the context of drug discovery, cryo-EM facilitates structure-based drug design by providing high-resolution structural data that can be used to identify binding sites for small molecules or biologics. For example, cryo-EM has been instrumental in elucidating the structures of G protein-coupled receptors (GPCRs) and ion channels, which are prominent drug targets. By visualizing the molecular interactions between these receptors and their ligands, researchers can gain insights into receptor modulation and pharmacology [17]. The ability to analyze these interactions at a molecular level is vital for the rational design of therapeutics.

Moreover, cryo-EM has shown promise in addressing previously "intractable" targets that are difficult to crystallize. For instance, it has been successfully applied to study integral membrane proteins, which represent a significant portion of druggable targets. The structural insights gained from cryo-EM studies have led to a better understanding of the lipid environment surrounding these proteins, further enhancing the accuracy of the structural data obtained [3].

Case studies illustrate the practical applications of cryo-EM in drug discovery. For instance, the structural determination of GPCR-G protein complexes has revealed critical information about drug-receptor interactions and the influence of accessory proteins on pharmacological responses [17]. Additionally, cryo-EM has been utilized to explore conformational diversity in proteins, which is essential for understanding how drugs can effectively modulate their activity [21].

In summary, cryo-EM advances protein structure determination by enabling high-resolution visualization of macromolecular complexes, thus providing essential insights for drug discovery. Its applications range from elucidating complex structures and understanding drug interactions to aiding in the design of novel therapeutics, thereby establishing cryo-EM as an indispensable tool in modern biomedical research. The ongoing developments in cryo-EM technology promise to further enhance its utility in structural biology and drug development, making it a cornerstone of contemporary pharmaceutical research [12].

5 Comparison with Traditional Methods ​

5.1 Cryo-EM vs. X-ray Crystallography ​

Cryo-electron microscopy (cryo-EM) has significantly advanced the field of protein structure determination, particularly in comparison to traditional methods such as X-ray crystallography. The evolution of cryo-EM technology has enabled researchers to study macromolecular structures at near-atomic resolution without the need for crystallization, which is often a major bottleneck in structural biology.

One of the primary advantages of cryo-EM is its ability to analyze large and complex biological assemblies that are difficult to crystallize. For instance, cryo-EM can probe proteins as small as hemoglobin (64 kDa) while circumventing the crystallization challenges entirely, which positions it as a complementary method to X-ray crystallography, particularly for larger, more flexible macromolecular complexes (Shoemaker & Ando, 2018) [18]. This is particularly important as traditional X-ray crystallography is often limited to well-ordered, smaller proteins and can struggle with dynamic or disordered structures.

Recent technological advancements in cryo-EM, including improvements in electron detection, imaging hardware, and software algorithms, have significantly enhanced the resolution of cryo-EM. It is now common to achieve resolutions of around 2 Å, allowing for the visualization of detailed atomic structures, a feat that was previously the domain of X-ray crystallography (Ognjenović et al., 2019) [6]. Furthermore, the development of direct electron detectors has led to unprecedented image quality, allowing for more accurate structural determination of proteins and their complexes (Bai et al., 2015) [22].

Cryo-EM also provides unique insights into the conformational states of proteins. While X-ray crystallography often captures a single static conformation, cryo-EM allows for the visualization of multiple conformations of a protein complex, thus providing a more comprehensive understanding of its functional dynamics (Cheng et al., 2015) [23]. The integration of cryo-EM data with other techniques, such as nuclear magnetic resonance (NMR) spectroscopy, further enhances the structural analysis by combining complementary data to provide a fuller picture of protein behavior (Geraets et al., 2020) [7].

Despite these advancements, cryo-EM does face certain limitations. The determination of structures for some biological targets at atomic resolution remains challenging, particularly for proteins with high intrinsic disorder or flexibility (Nwanochie & Uversky, 2019) [24]. Moreover, while cryo-EM has revolutionized the study of larger complexes, X-ray crystallography continues to excel in providing precise atomic coordinates for smaller, well-ordered macromolecules and can offer high-resolution dynamic information under varying conditions (Shoemaker & Ando, 2018) [18].

In summary, cryo-EM represents a transformative approach to protein structure determination, offering distinct advantages over traditional methods like X-ray crystallography, particularly in studying larger and more dynamic complexes. The ongoing advancements in cryo-EM technology promise to further enhance its capabilities, making it an essential tool in the structural biologist's toolkit.

5.2 Cryo-EM vs. NMR Spectroscopy ​

Cryo-electron microscopy (cryo-EM) has significantly advanced the field of protein structure determination, offering unique advantages over traditional methods such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. One of the most notable strengths of cryo-EM is its capability to visualize large and structurally heterogeneous protein complexes that are often difficult to analyze using conventional techniques. Cryo-EM can achieve near-atomic resolutions (better than 4 Å) and, in some cases, atomic resolutions (below 1.5 Å), making it a powerful tool for structural biology [2].

In contrast, X-ray crystallography typically requires the formation of well-ordered crystals, which can be a significant limitation when dealing with large or flexible proteins. Many proteins, especially those that are dynamic or membrane-bound, do not crystallize well, thus restricting the application of this technique [24]. NMR spectroscopy, while effective for small proteins, faces challenges with larger and more complex macromolecules due to limitations in size and the inherent flexibility of such proteins [24]. Cryo-EM, however, allows for the analysis of proteins in a near-native state without the need for crystallization, thus overcoming these barriers [3].

The methodological advancements in cryo-EM, such as improved instrumentation, direct electron detectors, and enhanced image processing algorithms, have led to substantial improvements in data quality and resolution. These advancements have facilitated the visualization of intricate protein structures and dynamic complexes that were previously inaccessible [6]. For example, cryo-EM has been successfully employed to resolve structures of integral membrane proteins, which represent a significant proportion of druggable targets, thereby enhancing drug discovery efforts [5].

When comparing cryo-EM with NMR spectroscopy, the former is particularly advantageous for studying large protein complexes and assemblies that exhibit significant conformational variability. NMR is limited to relatively small proteins and struggles with dynamic structures due to its reliance on the static conformations of molecules [24]. Cryo-EM can capture multiple conformations of a protein complex in a single experiment, providing insights into its functional dynamics [20].

Furthermore, the integration of cryo-EM data with NMR and other structural biology techniques can yield complementary information, allowing for a more comprehensive understanding of protein structures and dynamics [7]. This synergy can enhance the resolution and reliability of structural models, particularly for complex biological systems that exhibit intrinsic disorder or flexibility [24].

In summary, cryo-EM represents a transformative approach to protein structure determination, enabling researchers to tackle challenges that are often insurmountable with traditional methods like X-ray crystallography and NMR spectroscopy. Its ability to analyze large, flexible, and dynamic protein complexes in a near-native state positions cryo-EM as a critical tool in modern structural biology.

6 Future Perspectives in Cryo-EM ​

6.1 Potential Developments in Technology ​

Cryo-electron microscopy (cryo-EM) has significantly advanced the field of protein structure determination through a series of technological innovations and methodological improvements. This technique has evolved to become a powerful tool for elucidating the three-dimensional structures of biological macromolecules, offering unique advantages over traditional methods such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.

One of the key advancements in cryo-EM is the development of direct electron detection cameras and sophisticated image processing technologies. These innovations have enabled researchers to obtain structures of many important proteins at near-atomic resolution, and in some cases, at atomic resolution, thus overcoming previous challenges related to crystallization and low protein yield (Mio and Sato, 2018) [3]. For instance, the resolution achieved with single-particle cryo-EM has improved to approximately 2 Å for smaller proteins, while larger complexes have also been successfully analyzed (Ognjenović et al., 2019) [6].

Furthermore, cryo-EM allows for the visualization of protein complexes in their native cellular environment, which is particularly beneficial for studying dynamic and heterogeneous systems that may not crystallize well (Danev et al., 2019) [1]. The capability to capture multiple conformational states and reaction intermediates has expanded the structural landscape available to researchers, providing insights into the dynamics of protein function (Tsai et al., 2022) [19].

Looking towards the future, several potential developments in cryo-EM technology could further enhance its application in protein structure determination. These include improvements in sample preparation techniques that better mimic the lipid environment of membrane proteins, which have historically posed challenges in cryo-EM studies (Mio and Sato, 2018) [3]. Additionally, advancements in automated data acquisition and analysis software are expected to streamline workflows and increase the throughput of structure determination, thereby reducing the cost and skill barriers associated with the technique (Danev et al., 2019) [1].

Moreover, integrating cryo-EM with other structural biology techniques, such as NMR and X-ray crystallography, could lead to more comprehensive models of macromolecular complexes. This hybrid approach can provide complementary data that enhances the resolution and accuracy of structural models (Geraets et al., 2020) [7].

In summary, cryo-EM has revolutionized protein structure determination through significant technological advancements, enabling high-resolution analysis of complex biological systems. Future developments are poised to further enhance its capabilities, making it an even more integral tool in structural biology and drug design.

6.2 Expanding Applications in Structural Biology ​

Cryo-electron microscopy (cryo-EM) has significantly advanced the field of protein structure determination, offering unparalleled capabilities for resolving complex biological macromolecules at near-atomic resolution. Recent technological innovations, including improvements in microscope design, direct electron detectors, and advanced image processing software, have transformed cryo-EM into a mainstream technique in structural biology. These advancements allow for the visualization of protein structures that were previously inaccessible due to limitations in traditional methods like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.

One of the key advancements in cryo-EM is its ability to analyze proteins and protein complexes in their native, hydrated states, thus preserving their functional conformations. This feature is particularly advantageous for studying dynamic macromolecular assemblies, such as G protein-coupled receptors (GPCRs) and spliceosomes, which can undergo significant conformational changes during their biological activities (Ognjenović et al., 2019; Subramaniam et al., 2016). The integration of cryo-EM with other structural determination techniques, such as X-ray crystallography and NMR, enhances the overall understanding of complex biological systems by providing complementary information about static and dynamic structural states (Geraets et al., 2020).

The methodology of cryo-EM has evolved to include single-particle analysis (SPA) and cryo-electron tomography (cryo-ET), enabling the study of proteins as small as 100 kDa at resolutions approaching 2 Å (Ognjenović et al., 2019). SPA is particularly useful for determining high-resolution structures of isolated proteins, while cryo-ET allows for the visualization of macromolecular complexes in their native cellular environments, thereby offering insights into their functional states (Danev et al., 2019).

Future perspectives in cryo-EM suggest that continued methodological improvements will further enhance its reliability and throughput. These include refining sample preparation techniques, improving data acquisition protocols, and developing more sophisticated image processing algorithms. Such advancements are expected to lower the barriers for adoption and increase the accessibility of cryo-EM for a broader range of biological studies (Mio & Sato, 2018; Subramaniam et al., 2016).

Moreover, the application of cryo-EM in drug discovery is expanding, as it provides crucial structural insights into drug-target interactions at high resolutions. This capability is vital for the design of therapeutics targeting integral membrane proteins, which represent a significant portion of druggable targets (Lees et al., 2021). The ongoing integration of cryo-EM data with other biophysical techniques, such as molecular dynamics simulations, will enhance the understanding of protein flexibility and dynamics, ultimately leading to more effective drug design strategies (Torrens-Fontanals et al., 2020).

In summary, cryo-EM has revolutionized protein structure determination by enabling high-resolution visualization of macromolecular complexes in their native states. The future of cryo-EM appears promising, with ongoing technological advancements and an expanding range of applications in structural biology, including drug discovery and the study of dynamic biological processes. As these developments unfold, cryo-EM is poised to continue shaping the landscape of structural biology, offering deeper insights into the mechanisms of life at the molecular level.

7 Conclusion ​

Cryo-electron microscopy (cryo-EM) has emerged as a pivotal technology in the field of protein structure determination, revolutionizing the way researchers study complex biological macromolecules. The significant advancements in cryo-EM, particularly in detector technology, sample preparation methods, and image processing algorithms, have enhanced its capability to resolve structures at near-atomic and atomic resolutions. This has enabled the visualization of large, dynamic protein complexes that are often challenging to analyze using traditional methods like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Key findings from recent studies underscore cryo-EM's role in elucidating the structures of integral membrane proteins and macromolecular assemblies, which are crucial for drug discovery and understanding biological processes. The integration of cryo-EM with other structural biology techniques offers a comprehensive approach to studying protein dynamics and interactions. Looking ahead, continued technological innovations are expected to further expand the applications of cryo-EM, making it an indispensable tool in structural biology and therapeutic development. As the field progresses, cryo-EM is poised to uncover new insights into the molecular mechanisms underlying health and disease, ultimately contributing to advancements in biomedical research and drug design.

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