Exploring icosahedral virus structures with VIPER

Reconstruction principles of icosahedral virus structure determination using electron cryomicroscopy

Cryo-electron microscopy (cryo-EM), the structural analysis of samples embedded in vitreous ice, is a powerful approach for determining three-dimensional (3D) structures of biological specimens. Over the past two decades, this technique has been used to successfully calculate subnanometer (<10 Å) resolution and, in some cases, near-atomic resolution structures of highly symmetrical and stable complexes such as icosahedral viruses and ribosomes, as well as samples that form ordered two-dimensional or helical arrays. However, determining high-resolution 3D structures of smaller, less symmetrical, and dynamic samples remains a significant challenge. The recent development of electron microscopes with automated data collection capabilities and robust direct electron detection cameras, as well as new powerful image processing algorithms, has dramatically expanded the number of biological macromolecules amenable for study using cryo-EM. In addition, these new technological and computational developments have been used to successfully determine <5 Å resolution 3D structures of samples, such as membrane proteins and complexes with either low or no symmetry, that traditionally were not considered promising candidates for high-resolution cryo-EM. With these exciting new advances, cryo-EM is now on pace to determine atomic resolution 3D structures.

Editorial overview: New protein production tools for structural biology

SAXS Ensemble Refinement of ESCRT-III CHMP3 Conformational Transitions

Single particle analysis integrated with microscopy: A high-throughput approach for reconstructing icosahedral particles

The cryo-EM structure of ETB-Gi complex, obtained using the Fusion-G system, provides insights into the distinct activation mechanism and G-protein promiscuity of ETB, aiding in the design of ETB agonists for therapeutic applications.

The structure of a T = 1 icosahedral particle (where T is the triangulation number), assembled from southern bean mosaic virus coat protein fragments that lacked the amino-terminal arm, was solved by means of model building procedures with the use of 6-angstrom resolution x-ray diffraction data. The icosahedral five-, three-, and twofold contacts were found to be similar, at this resolution, to the analogous contacts (icosahedral five-, quasi-three-, and quasi-twofolds) found in the parent T = 3 southern bean mosaic virus. However, the icosahedral fivefold contacts of the T = 3 structure are the most conserved in the T = 1 capsid. These results are consistent with a mechanism in which pentameric caps of dimers are the building blocks for the assembly of T = 1 and T = 3 icosahedral viruses.

We discuss recent advancements in structural biology methods for investigating sites of protein-protein interactions. We will inform readers outside the field of structural biology about techniques beyond crystallography, and how these different technologies can be utilized for drug development. Advancements in cryo-electron microscopy (cryoEM) and micro-electron diffraction (microED) may change how we view atomic resolution structural biology, such that well-ordered macrocrystals of protein complexes are not required for interface identification. However, some drug discovery applications, such as lead peptide compound generation, may not require atomic resolution; mass spectrometry techniques can provide an expedited path to generation of lead compounds. New crosslinking compounds, more user-friendly data analysis, and novel protocols such as protein painting can advance drug discovery programs, even in the absence of atomic resolution structural data. Finally, artificial intelligence and machine learning methods, while never truly replacing experimental methods, may provide rational ways to stratify potential druggable regions identified with mass spectrometry into higher and lower priority candidates. Electron diffraction of nanocrystals combines the benefits of both x-ray diffraction and cryoEM, and may prove to be the next generation of atomic resolution protein-protein interface identification. However, in situations such as peptide drug discovery, mass spectrometry techniques supported by advancements in computational methods will likely prove sufficient to support drug discovery efforts. In addition, these methods can be significantly faster than any crystallographic or cryoEM methods for identification of interacting regions.

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.

Aura and Sindbis viruses are closely related alphaviruses. Unlike other alphaviruses, Aura virus efficiently encapsidates both genomic RNA (11.8 kb) and subgenomic RNA (4.2 kb) to form virus particles. Previous studies on negatively stained Aura virus particles predicted that there were two major size classes with potential T=3 and T=4 capsid structures. We have used cryoelectron microscopy and three-dimensional image reconstruction techniques to examine the native morphology of different classes of Aura virus particles produced in BHK cells. Purified particles separated into two components in a sucrose gradient. Reconstructions of particles in the top and bottom components were computed to resolutions of 17 and 21 A, respectively, and compared with reconstructions of Sindbis virus and Ross River virus particles. Aura virus particles of both top and bottom components have similar, T=4 structures that resemble those of other alphaviruses. The morphology of Aura virus glycoprotein spikes closely resembles that of Sindbis virus spikes and is detectably different from that of Ross River virus spikes. Thus, some aspects of the surface structure of members of the Sindbis virus lineage have been conserved, but other aspects have diverged from the Semliki Forest/Ross River virus lineage.

After budding, the human immunodeficiency virus (HIV) must 'mature' into an infectious viral particle. Viral maturation requires proteolytic processing of the Gag polyprotein at the matrix-capsid junction, which liberates the capsid (CA) domain to condense from the spherical protein coat of the immature virus into the conical core of the mature virus. We propose that upon proteolysis, the amino-terminal end of the capsid refolds into a beta-hairpin/helix structure that is stabilized by formation of a salt bridge between the processed amino-terminus (Pro1) and a highly conserved aspartate residue (Asp51). The refolded amino-terminus then creates a new CA-CA interface that is essential for assembling the condensed conical core. Consistent with this model, we found that recombinant capsid proteins with as few as four matrix residues fused to their amino-termini formed spheres in vitro, but that removing these residues refolded the capsid amino-terminus and redirected protein assembly from spheres to cylinders. Moreover, point mutations throughout the putative CA-CA interface blocked capsid assembly in vitro, core assembly in vivo and viral infectivity. Disruption of the conserved amino-terminal capsid salt bridge also abolished the infectivity of Moloney murine leukemia viral particles, suggesting that lenti- and oncoviruses mature via analogous pathways.

Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy

Integrins are a large family of heterodimeric transmembrane signaling proteins that affect diverse biological processes such as development, angiogenesis, wound healing, neoplastic transformation, and thrombosis. We report here the three-dimensional structure at 20-A resolution of the unliganded, low-affinity state of the human platelet integrin alpha(IIb)beta(3) derived by electron cryomicroscopy and single particle image reconstruction. The large ectodomain and small cytoplasmic domains are connected by a rod of density that we interpret as two parallel transmembrane alpha-helices. The docking of the x-ray structure of the alpha(V)beta(3) ectodomain into the electron cryomicroscopy map of alpha(IIb)beta(3) requires hinge movements at linker regions between domains in the crystal structure. Comparison of the putative high- and low-affinity conformations reveals dramatic conformational changes associated with integrin activation.

We have described four superfamilies of enzymes where the members within each catalyze different overall reactions using broadly varied substrates. Within each superfamily, the different overall reactions are facilitated by a common mechanistic strategy that can be rationalized in the context of the structural scaffold. Although space limitations prevent their discussion, a number of additional superfamilies have been described in which these principles appear to obtain. Analyses of the relationships between structure and function in all of these superfamilies suggest two general conclusions regarding the evolution of new catalytic activities.

Relationships between RNA topology and nucleocapsid structure in a model icosahedral virus