Cryo-EM in Enzymology

<p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="first" dir="auto" id="d20682e98">Cryo-electron microscopy (Cryo-EM) has emerged as a powerful tool for determining the structures of biomolecules, especially in cases where traditional methods like X-ray crystallography and nuclear magnetic resonance (NMR) fall short [ <a class="xref-link" href="#r1">1</a>]. Its ability to visualize biomolecules in near-native states at cryogenic temperatures has revolutionized structural biology, leading to the resolution of complex molecular machines such as the ribosome and membrane proteins, which are critical targets for drug development. The recent “resolution revolution” [ <a class="xref-link" href="#r2">2</a>] in Cryo-EM has made it possible to achieve atomic or near-atomic resolution for many large macromolecular assemblies. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d20682e106">In Cryo-EM, a substantial volume of particle data is often excluded due to inadequate phase contrast, which impedes full utilization of these particles. This limited insight into discarded structures constrains our capacity to attain the utmost resolution. Furthermore, the 2D classification task in single-particle reconstruction proves especially challenging for heterogeneous samples, low-concentration or low-symmetry structures, and small (&lt;250 kDa) or flexible molecules. To enhance phase contrast, a conventional approach involves integrating a phase-shifting device—such as Zernike or Volta phase plates—within the back focal plane of the objective lens. However, these devices are prone to high-frequency signal attenuation, variability in fabrication quality, reliability issues, and shortened operational lifespans due to electrostatic charging. In recent years, computational phase retrieval algorithms have advanced markedly and are now widely applied in coherent diffraction imaging, holographic imaging, and Fourier ptychographic microscopy. Some studies in Cryo-EM have also begun investigating the potential of computational phase retrieval to extend imaging contrast to the limits allowable by our understanding of the physics of contrast formation [ <a class="xref-link" href="#r3">3</a>].

The Chikungunya virus is carried by mosquitos and can cause a number of diseases in humans including encephalitis, which can be fatal in some cases, and severe arthritis. A recent mutation in the E1 protein of the virus has allowed it to efficiently reproduce in a different species of mosquitos, leading to a Chikungunya epidemic in Réunion Island in 2005 and the subsequent infection of millions of individuals in Africa and Asia. The virus also has the potential to spread to many areas of Europe and the Americas. Chikungunya virus has a single-stranded RNA genome that codes for four non-structural proteins and five structural proteins. Based on this knowledge it has been possible to develop virus-like particles that can be used to immunise non-human primates against Chikungunya infection by inducing antibody production. However, the development of vaccines for Chikungunya in humans will require a deeper understanding of how these antibodies produced by the vaccine interact with the virus and more detailed information about the structures of the virus and antibodies. Sun et al. have used two techniques – X-ray crystallography and electron cryo-microscopy – to determine the structure of Chikungunya virus-like particles, and to obtain new insights into the interactions of these particles with four related antibodies. Electron cryo-microscopy was used to figure out the structure of the particles at near atomic resolution, and X-ray crystallography was used to determine the atomic resolution structures of two of the four Fab antibodies that neutralize the Chikungunya virus. Electron cryo-microscopy was also used to probe the complex formed by the interactions between the virus-like particles and the antibodies. Sun et al. were able to identify the likely viral receptor site that is blocked by three of the antibodies when they neutralize the virus; the fourth antibody is thought to act by immobilizing one of the domains of protein E2, thereby hiding the “fusion loop” that allows the virus to enter and infect human tissue. It is hoped that these findings will contribute to efforts to combat the spread of the Chikungunya virus worldwide.

Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) have transformed structural biology by enabling the visualization of biological macromolecules at near-atomic resolution. Since its establishment in 2002, the Electron Microscopy Data Bank (EMDB) has played a pivotal role in this “Resolution Revolution,” serving as a central repository for cryo-EM data. Over the past decade, major advances in detector technology, image processing, and analytical methodologies have led to a surge in cryo-EM structure depositions. This study provides a comprehensive analysis of temporal trends in the EMDB, highlighting the growing number of entries, improvements in resolution, and the evolution of detector usage. It emphasizes the rise of single-particle analysis (SPA) as the dominant cryo-EM technique and examines how high-resolution detectors have enhanced data quality. Additionally, it explores resolution standards across leading scientific journals, underscoring the role of EMDB not only as a data archive but also as a catalyst for innovation in structural biology.

In modern structural biology, there are three major methods for structural biologists to obtain structural information of macromolecules: cryo-electron microscopy (cryo-EM), nuclear magnetic resonance (NMR), and X-ray crystallography. Cryo-EM, in comparison with the other two methods, allows structural biologists to obtain the structures of various macromolecules in a more native and less perturbed system. Over the past decade, cryo-EM has enabled scientists to determine the structures of protein complexes at atomic resolution and made a profound impact in molecular bioscience and pharmaceutical sectors. Along with cryo-EM, another emerging technique called cryo-electron tomography (cryo-ET) has gained increasing importance in structural biology. It has the potential to visualize macromolecular complexes and assemblies in their native environments at high resolution, but there are still some challenges for small, sparse subjects and in approaching atomic resolution in situ. This chapter summarizes the major steps involved in structure determination using cryo-EM and cryo-ET and highlights the major challenges for in situ cryo-ET. We also present a few examples of near-atomic resolution structure determination of macromolecular assemblies both in purified systems in vitro and in native contexts in situ. Future perspectives are discussed as well.

Cryo-electron microscopy (cryo-EM) has transformed structural biology by enabling the determination of macromolecular structures at near-atomic resolution. The recent Nobel Prizes awarded to John Hopfield and Geoffrey Hinton in Physics, and to David Baker, Demis Hassabis, and John Jumper in Chemistry, highlight the growing convergence of artificial intelligence (AI) and computational innovation in advancing scientific discovery. Cryo-EM has emerged as a powerful tool for elucidating the architectures and dynamics of complex biological assemblies. Its ability to capture multiple conformational states provides mechanistic insights that are critical for understanding molecular function. However, traditional cryo-EM data processing pipelines face challenges in handling large, heterogeneous datasets. Manual particle selection, extensive computational demands, and uneven sampling of conformational states often limit resolution, especially for rare or transient conformations. Recent advances in AI-driven methods are reshaping this landscape. Deep neural networks now facilitate automated particle picking, denoising, classification, and three-dimensional reconstruction, substantially reducing human bias and computational bottlenecks. Machine learning models have also enhanced the accuracy of map interpretation and atomic modeling, accelerating the transition from raw micrographs to high-resolution structures. These developments represent a paradigm shift, positioning AI as an integral component of the “resolution revolution” in structural biology. This comprehensive article examines the state of the art in cryo-electron microscopy, highlighting how artificial intelligence is redefining every stage of the workflow-from automated particle picking and map reconstruction to structure modeling and high-resolution refinement. The integration of deep learning and neural network-based tools has transformed cryo-EM into a data-driven and highly precise discovery platform. By showcasing these innovations, we emphasize AI’s pivotal role in advancing next-generation cryo-EM research and accelerating molecular insights with profound implications for structural biology and drug discovery. Not applicable.

Structural studies of biocomplexes using single-particle cryo-electron microscopy (cryo-EM) is now a well-established technique in structural biology and has become competitive with X-ray crystallography. The latest advances in EM enable us to determine structures of protein complexes at 3-5 Å resolution for an extremely broad range of sizes from ~200 kDa up to hundreds of megadaltons (Bartesaghi et al., Science 348(6239):1147-1151, 2051; Bai et al., Nature 525(7568):212-217, 2015; Vinothkumar et al., Nature 515(7525):80-84, 2014; Grigorieff and Harrison, Curr Opin Struct Biol 21(2):265-273, 2011). The majority of biocomplexes comprise a number of different components and are not amenable to crystallisation. Secretion systems are typical examples of such multi-protein complexes, and structural studies of them are extremely challenging. The only feasible approach to revealing their spatial organisation and functional modification is cryo-EM. The development of systems for digital registration of images and algorithms for the fast and efficient processing of recorded images and subsequent analysis facilitated the determination of structures at near-atomic resolution. In this review we will describe sample preparation for cryo-EM, how data are collected by new detectors, and the logistics of image analysis through the basic steps required for reconstructions of both small and large biological complexes and their refinement to nearly atomic resolution. The processing workflow is illustrated using examples of EM analysis of a Type IV Secretion System.

Recent developments in cryo-electron microscopy (cryo-EM) hardware along with continuously evolving software tools have led to the discovery of many novel structures that it was not possible to solve until now, resulting in what is termed “the resolution revolution”. In structural virology, it has also led to a re-evaluation of known structures. Most virion structures solved by X-ray crystallography or cryo-EM are focused on the capsid protein (CP) as a result of the application of icosahedral symmetry averaging to “improve” the electron density maps. However, this has the consequence that the intrinsic asymmetry of important components of virions, such as the viral genome and structural proteins lacking such symmetry, are masked. Single-stranded (ss), positive-sense RNA viruses are major pathogens in all kingdoms of life. Asymmetric cryo-EM structure determination of a major model virus in this class, bacteriophage MS2, reveals the limitations of a symmetrized view. As well as the presence and interactions made by the unique Maturation Protein, it also reveals multiple gRNA-CP dimer contacts corresponding to our previous prediction that dispersed, sequence-degenerate RNA motifs (Packaging Signals, PSs) play important roles during the virion assembly. Here, we describe how relaxing symmetry during structure determination can image such gRNA-PS contacts in a range of ssRNA viruses including the picornavirus Bovine Enterovirus-1, the alphaviruses Sindbis and Semliki Forest Viruses, as well as the plant virus Turnip Crinkle Virus. The revelation of these functionally important gRNA-CP contacts changes our fundamental understanding of assembly in these pathogens and may have further translational importance.

Analysis of RNA structure using small-angle X-ray scattering

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the nucleotide-free state of Gα.

The rapidly evolving role of cryo-EM in drug design

Cryo-transmission electron microscopy (Cryo-TEM) and particularly single particle analysis is rapidly becoming the premier method for determining the three-dimensional structure of protein complexes, and viruses. In the last several years there have been dramatic technological improvements in Cryo-TEM, such as advancements in automation and use of improved detectors, as well as improved image processing techniques. While Cryo-TEM was once thought of as a low resolution structural technique, the method is currently capable of generating nearly atomic resolution structures on a routine basis. Moreover, the combination of Cryo-TEM and other methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics modeling are allowing researchers to address scientific questions previously thought intractable. Future technological developments are widely believed to further enhance the method and it is not inconceivable that Cryo-TEM could become as routine as X-ray crystallography for protein structure determination.

Low cost, high performance GPU computing solution for atomic resolution cryoEM single-particle reconstruction

High-resolution electron cryomicroscopy of macromolecular assemblies

Hybrid structure fitting methods combine data from cryo-electron microscopy and X-ray crystallography with molecular dynamics simulations for the determination of all-atom structures of large biomolecular complexes. Evaluating the quality-of-fit obtained from hybrid fitting is computationally demanding, particularly in the context of a multiplicity of structural conformations that must be evaluated. Existing tools for quality-of-fit analysis and visualization have previously targeted small structures and are too slow to be used interactively for large biomolecular complexes of particular interest today such as viruses or for long molecular dynamics trajectories as they arise in protein folding. We present new data-parallel and GPU-accelerated algorithms for rapid interactive computation of quality-of-fit metrics linking all-atom structures and molecular dynamics trajectories to experimentally-determined density maps obtained from cryo-electron microscopy or X-ray crystallography. We evaluate the performance and accuracy of the new quality-of-fit analysis algorithms vis-à-vis existing tools, examine algorithm performance on GPU-accelerated desktop workstations and supercomputers, and describe new visualization techniques for results of hybrid structure fitting methods.

Electron cryomicroscopy (cryo-EM) is a powerful tool in structural biology for determining 3D structures of macromolecular complexes. In the previous decade, many technological advancements have been introduced to improve the methodology of single particle cryo-EM, and hence the resolution of its protein reconstructions. These advancements include developments in both software algorithms and microscope hardware. Despite all these developments, the resolution of single particle cryo-EM cannot surpass 1.5 Å, a barrier blocking direct visualization of atomic details of the protein structure. Here, we show that by improving the optical performance of the electron microscope with new hardware, such resolution barrier can be removed, and atomic resolution of 1.25 Å can be reached with an engineered protein apoferritin. The atomic resolution protein structure helped set benchmarks for validating data quality in high resolution cryo-EM. We found that the new microscope also benefited to other proteins with lower contrast and higher flexibility, such as proteasome, chaperonin and fatty acid synthetase. For these proteins, sample properties and behavior were the major limitations. We anticipate that this would be alleviated by introducing other new microscope hardware, so that in the future, time and effort of structural biologists can concentrate on biochemical preparation of the sample, and single particle cryo-EM can routinely yield high quality protein structures at atomic resolution.