Jaekyung Hyun

During poxvirus morphogenesis, the external scaffold defines the shape and size of immature virions. However, its structural characterization has been hindered by the pleomorphic nature of authentic virus particles. Here, we present single-particle cryo-electron microscopy and cryo-electron tomography structures of two distinct in vitro assemblies of the vaccinia virus scaffold protein D13 that recapitulate oligomeric states previously observed in situ. These structures reveal a dramatic transition from rod-like oligomers, representing a pre-assembly state, to a fully formed scaffold, triggered by the docking of the N-terminal peptide of the viral membrane protein A17 into the base cavity of D13. We propose that this interaction destabilizes the pre-assembly conformation and initiates scaffold formation upon viral membrane recruitment, ultimately leading to the formation of immature virions. Our findings provide structural insights into the early stages of poxvirus morphogenesis and establish a mechanistic link between conformational changes in D13 and scaffold assembly.

Many archaea swim by means of rotating helical filaments called archaella. Most archaella are about 10 nm in diameter and comprise multiple copies of the protein archaellin. Here, we describe two archaellum structures formed by the ArlA2 or ArlB archaellins from the haloarchaeon Haloarcula marismortui. We found that both filaments have an additional proteinaceous outer sheath surrounding their inner core, a feature not observed previously in archaea. The outer sheath structures of the two filaments differ fundamentally. The outer domain of ArlB archaellin rotates by 180^°, forming stable dimers that likely increase the filament rigidity. In contrast, neither rotation nor dimerization of the outer domain was observed in ArlA2 filaments. 3D variability analysis demonstrated that the motions of ArlA2 and ArlB filaments are significantly distinct. Additionally, the ArlB filament displays a larger negatively charged surface area than ArlA2, which may help adaptation to higher salt concentrations. Our structural findings provide insight into how the two filaments can adapt to their different environments. Larger archaellins with additional outer domains can be found in various groups of archaea. We propose that such proteins may allow hosts to modify the properties of their archaella to enhance environmental adaptation.

G proteins and arrestins are key transducers for G protein-coupled receptor (GPCR) signaling, mediating distinct downstream pathways. Recent evidence suggests that G proteins and β-arrestins (βarrs) can directly or functionally interact. However, the molecular details and functional consequences of Gα–βarr interactions remain poorly understood. Here, we quantify the binding affinities between βarr1 and Gαs or Gαi1 in various activation states using microscale thermophoresis (MST). βarr1 in the active conformational ensemble state favors binding, whereas Gα activation status is less determinant. Hydrogen/deuterium exchange mass spectrometry reveals distinct conformational changes between Gαs versus Gαi1 upon βarr1 binding, suggesting differential binding mechanism between Gαs–βarr1 and Gαi1–βarr1 complexes. Both the Ras-like domain and the α-helical domain of Gα contribute to complex formation. Functionally, a BODIPY-FL–GTPγS assay shows that βarr1 does not alter GDP/GTP turnover of Gαs or Gαi1, whereas β-strand XX (βXX) release assays demonstrate that Gαs enhances βarr1 C-tail release. Together, these results propose molecular mechanism of the interaction and asymmetric functional coupling within Gα–βarr complexes and uncover a previously underappreciated layer of GPCR signal transduction.

During poxvirus morphogenesis, the external scaffold plays a key role in defining the shape and size of immature virions. However, its structural characterization has been hindered by the pleomorphic nature of authentic virus particles. Here, we present single-particle cryo-electron microscopy and cryo-electron tomography structures of two distinct in vitro assemblies of the vaccinia virus scaffold protein D13 that recapitulate oligomeric states previously observed in situ. These structures reveal a dramatic transition from rod-like oligomers, representing a pre-assembly state, to a fully formed scaffold, triggered by the docking of N-terminal peptide of the membrane protein A17 into the base cavity of D13. We propose that this interaction destabilizes the pre-assembly conformation and initiates scaffold formation upon viral membrane recruitment, ultimately leading to the immature virion. Our findings provide novel structural insights into the early stages of poxvirus morphogenesis and establish a mechanistic link between conformational changes in D13 and scaffold assembly.

Coliphage N4 is a representative species of the Schitoviridae family of bacteriophages. Originally structurally studied in 2008, the capsid structure was solved to 14 Å to reveal an interesting arrangement of Ig-like decoration proteins across the surface of the capsid. Herein, we present a high-resolution N4 structure, reporting a 2.45 Å map of the capsid obtained via single particle cryogenic-electron microscopy. Structural analysis of the major capsid proteins (MCPs) and decoration proteins (gp56 and gp17) of phage N4 reveals a pattern of interactions across the capsid that are mediated by structurally homologous domains of gp17. In this study, an analysis of the complex interface contacts allows us to confirm that the gp17 Ig-like decoration proteins of N4 are likely employed by the virus to increase the capsid’s structural integrity.

Poxviruses are double-stranded DNA viruses that represent the largest known highly pathogenic viruses infecting humans. They undergo dramatic morphological changes during their maturation process, resulting in structural differences between each virion, and their surface is decorated with more than a dozen randomly distributed surface proteins that facilitate viral entry. These are the main reasons poxviruses have eluded high-resolution structure determination. Over the last three decades, cryo-EM has developed into a mature technology that can increasingly overcome such problems of structural heterogeneity through advances in microscope technology and image processing algorithms. Here, we discuss the essential current modalities in cryo-EM, which promise to solve the structure of poxviruses in parts and as entire virions at near-atomic resolution. With a focus on cryo modalities, we provide an overview of methods, including volume microscopy by plasma ion beam milling, focused ion beam lamella preparation, subtomogram averaging, and single particle averaging. Protocols for poxvirus propagation, purification, and imaging by cryo-EM are presented. This chapter is aimed at experts and nonexpert researchers to help facilitate entry into the structural biology of this critical field in virology.

Podophages that infect gram-negative bacteria, such as Pectobacterium pathogen ΦM1, encode tail assemblies too short to extend across the complex gram-negative cell wall. To overcome this, podophages encode a large protein complex (ejectosome) packaged inside the viral capsid and correspondingly ejected during infection to form a transient channel that spans the periplasmic space. Here we describe the ejectosome of bacteriophage ΦM1 to a resolution of 3.32 Å by single particle cryo-EM. The core consists of tetrameric and octameric ejection proteins which form a ∼1.5 MDa ejectosome that must transition through the ∼30 Å aperture created by the short tail nozzle assembly that acts as the conduit for the passage of DNA during infection. The ejectosome forms several grooves into which coils of genomic DNA are fit before the DNA sharply turns and goes down the tunnel and into the portal. In addition, we reconstructed the icosahedral capsid and hybrid tail apparatus to resolutions between 3.04 Å and 3.23 Å, and note an uncommon fold adopted by the dimerized decoration proteins which further emphasize the structural diversity of podophages. These reconstructions have allowed the generation of a complete atomic model of the ΦM1, uncovering two distinct decoration proteins and highlighting the exquisite structural diversity of tailed bacteriophages.

Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure‐guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR‐associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo‐electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure‐guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.