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Human C-reactive protein (CRP) is a pentameric complex involved in immune defense and regulation of autoimmunity. CRP is also a therapeutic target, with both administration and depletion of serum CRP being pursued as a possible treatment for autoimmune and cardiovascular diseases, among others. CRP binds to phosphocholine (PC) moieties on membranes to activate the complement system via the C1 complex, but it is unknown how CRP, or any pentraxin, binds to C1. Here, we present a cryoelectron tomography (cryoET)-derived structure of CRP bound to PC ligands and the C1 complex. To gain control of CRP binding, a synthetic mimotope of PC was synthesized and used to decorate cell-mimetic liposome surfaces. Structure-guided mutagenesis of CRP yielded a fully active complex able to bind PC-coated liposomes that was ideal for cryoET and subtomogram averaging. In contrast to antibodies, which form Fc-mediated hexameric platforms to bind and activate the C1 complex, CRP formed rectangular platforms assembled from four laterally associated CRP pentamers that bind only four of the six available globular C1 head groups. Potential residues mediating lateral association of CRP were identified from interactions between unit cells in existing crystal structures, which rationalized previously unexplained mutagenesis data regarding CRP-mediated complement activation. The structure also enabled interpretation of existing biochemical data regarding interactions mediating C1 binding and identified additional residues for further mutagenesis studies. These structural data therefore provide a possible mechanism for regulation of complement by CRP, which limits complement progression and has consequences for how the innate immune system influences autoimmunity. [ABSTRACT FROM AUTHOR]

The revolutionary technique cryoelectron tomography (cryo-ET) enables imaging of cellular structure and organization in a near-native environment at submolecular resolution, which is vital to subsequent data analysis and modeling. The conventional structure detection process first reconstructs the three-dimensional (3D) tomogram from a series of two-dimensional (2D) projections and then directly detects subcellular components found within the tomogram. However, this process is challenging due to potential structural information loss during the tomographic reconstruction and the limited scope of existing methods since most major state-of-the-art object detection methods are designed for 2D rather than 3D images. Therefore, in this article, as an alternative approach to complement the conventional process, we propose a novel 2D-to-3D framework that detects structures within 2D projection images before reconstructing the results back to 3D. We implemented the proposed framework as three specific algorithms for three individual tasks: semantic segmentation, edge detection, and object localization. As experimental validation of the 2D-to-3D framework for cryo-ET data, we applied the algorithms to the segmentation of mitochondrial calcium phosphate granules, detection of spherical edges, and localization of mitochondria. Quantitative and qualitative results show better performance for prediction tasks of segmentation on the 2D projections and promising performance on object localization and edge detection, paving the way for future studies in the exploration of cryo-ET for in situ structural biology.

The HIV-1 capsid houses the viral genome and interacts extensively with host cell proteins throughout the viral life cycle. It is composed of capsid protein (CA), which assembles into a conical fullerene lattice composed of roughly 200 CA hexamers and 12 CA pentamers. Previous structural analyses of individual CA hexamers and pentamers have provided valuable insight into capsid structure and function, but detailed structural information about these assemblies in the broader context of the capsid lattice is lacking. In this study, we combined cryoelectron tomography and single particle analysis (SPA) cryoelectron microscopy to determine structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also developed a method of liposome scaffold-based in vitro lattice assembly ("lattice templating") that enabled us to directly study the lattice under a wider range of conditions than has previously been possible. Using this approach, we identified a critical role for inositol hexakisphosphate in pentamer formation and determined the structure of the CA lattice bound to the capsid-targeting antiretroviral drug GS-6207 (lenacapavir). Our work reveals key structural details of the mature HIV-1 CA lattice and establishes the combination of lattice templating and SPA as a robust strategy for studying retroviral capsid structure and capsid interactions with host proteins and antiviral compounds. [ABSTRACT FROM AUTHOR]

Cryoelectron tomography directly visualizes heterogeneous macromolecular structures in their native and complex cellular environments. However, existing computerassisted structure sorting approaches are low throughput or inherently limited due to their dependency on available templates and manual labels. Here, we introduce a high-throughput template-and-label-free deep learning approach, Deep Iterative Subtomogram Clustering Approach (DISCA), that automatically detects subsets of homogeneous structures by learning and modeling 3D structural features and their distributions. Evaluation on five experimental cryo-ET datasets shows that an unsupervised deep learning based method can detect diverse structures with a wide range of molecular sizes. This unsupervised detection paves the way for systematic unbiased recognition of macromolecular complexes in situ. [ABSTRACT FROM AUTHOR]

The Porphyromonas gingivalis type IX secretion system (T9SS) promotes periodontal disease by secreting gingipains and other virulence factors. By in situ cryoelectron tomography, we report that the P. gingivalis T9SS consists of 18 PorM dimers arranged as a large, caged ring in the periplasm. Near the outer membrane, PorM dimers interact with a PorKN ring complex of ∼52 nm in diameter. PorMKN translocation complexes of a given T9SS adopt distinct conformations energized by the proton motive force, suggestive of different activation states. At the inner membrane, PorM associates with a cytoplasmic complex that exhibits 12-fold symmetry and requires both PorM and PorL for assembly. Activated motors deliver substrates across the outer membrane via one of eight Sov translocons arranged in a ring. The T9SSs are unique among known secretion systems in bacteria and eukaryotes in their assembly as supramolecular machines composed of apparently independently functioning translocation motors and export pores. [ABSTRACT FROM AUTHOR]

F plasmids circulate widely among the Enterobacteriaceae through encoded type IV secretion systems (T4SSFs). Assembly of T4SSFs and associated F pili requires 10 VirB/VirD4‐like Tra subunits and eight or more F‐specific subunits. Recently, we presented evidence using in situ cryoelectron tomography (cryoET) that T4SSFs undergo structural transitions when activated for pilus production, and that assembled pili are deposited onto alternative basal platforms at the cell surface. Here, we deleted eight conserved F‐specific genes from the MOBF12C plasmid pED208 and quantitated effects on plasmid transfer, pilus production by fluorescence microscopy, and elaboration of T4SSF structures by in situ cryoET. Mutant phenotypes supported the assignment of F‐specific subunits into three functional Classes: (i) TraF, TraH, and TraW are required for all T4SSF‐associated activities, (ii) TraU, TraN, and TrbC are nonessential but contribute significantly to distinct T4SSF functions, and (iii) TrbB is essential for F pilus production but not for plasmid transfer. Equivalent mutations in a phylogenetically distantly related MOB12A F plasmid conferred similar phenotypes and generally supported these Class assignments. We present a new structure‐driven model in which F‐specific subunits contribute to distinct steps of T4SSF assembly or activation to regulate DNA transfer and F pilus dynamics and deposition onto alternative platforms. [ABSTRACT FROM AUTHOR]

Bidirectional transport in cilia is carried out by polymers of the IFTA and IFTB protein complexes, called anterograde and retrograde intraflagellar transport (IFT) trains. Anterograde trains deliver cargoes from the cell to the cilium tip, then convert into retrograde trains for cargo export. We set out to understand how the IFT complexes can perform these two directly opposing roles before and after conversion. We use cryoelectron tomography and in situ cross-linking mass spectrometry to determine the structure of retrograde IFT trains and compare it with the known structure of anterograde trains. The retrograde train is a 2-fold symmetric polymer organized around a central thread of IFTA complexes. We conclude that anterograde-to-retrograde remodeling involves global rearrangements of the IFTA/B complexes and requires complete disassembly of the anterograde train. Finally, we describe how conformational changes to cargo-binding sites facilitate unidirectional cargo transport in a bidirectional system. [Display omitted] • Cryo-ET structure of retrograde IFT trains • IFTA forms the core and makes symmetrical polymeric interfaces • IFT train remodeling at the tip involves complete disassembly of anterograde train • Remodeling generates unique cargo-binding surfaces Visualizing retrograde IFT trains by in situ cryoelectron tomography and structural proteomics reveals how trains are remodeled at the cilium tip and provides insight into the regulation of selective cargo transport. [ABSTRACT FROM AUTHOR]

Mammalian oocytes are filled with poorly understood structures called cytoplasmic lattices. First discovered in the 1960s and speculated to correspond to mammalian yolk, ribosomal arrays, or intermediate filaments, their function has remained enigmatic to date. Here, we show that cytoplasmic lattices are sites where oocytes store essential proteins for early embryonic development. Using super-resolution light microscopy and cryoelectron tomography, we show that cytoplasmic lattices are composed of filaments with a high surface area, which contain PADI6 and subcortical maternal complex proteins. The lattices associate with many proteins critical for embryonic development, including proteins that control epigenetic reprogramming of the preimplantation embryo. Loss of cytoplasmic lattices by knocking out PADI6 or the subcortical maternal complex prevents the accumulation of these proteins and results in early embryonic arrest. Our work suggests that cytoplasmic lattices enrich maternally provided proteins to prevent their premature degradation and cellular activity, thereby enabling early mammalian development. [Display omitted] • Oocytes store essential proteins for early embryogenesis on cytoplasmic lattices • Cytoplasmic lattices are composed of periodic filaments with high surface area • Many stored proteins are required for epigenetic reprogramming of the embryo • Study links infertility phenotypes in women with stored protein function Mammalian oocytes enrich proteins needed to sustain early embryonic development on cytoplasmic lattices. Loss of cytoplasmic lattices leads to reduced levels of maternally supplied proteins and embryogenesis failure. [ABSTRACT FROM AUTHOR]

Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle. [Display omitted] • Cryoelectron tomography enabled characterization of rotavirus assembly intermediates • Subtomogram averaging provided detailed descriptions of the intermediates • Tri-lobed conformation of intact attachment protein VP4 described in enveloped stage • RNA-dependent RNA polymerase, VP1, has a role in recruiting genomes to particles Shah et al. used cryo-tomography to characterize the assembly pathway of rotavirus inside infected cells. The use of flash frozen samples preserved the assembly stages in their native state and relative proportions, and subtomogram averaging revealed their molecular structures, to near atomic detail in the best case. [ABSTRACT FROM AUTHOR]

Cryoelectron tomography is a method to image biological samples three-dimensionally at molecular resolution. This modality provides insights into intracellular processes in their physiological settings. Obtaining a high-quality sample for cryoelectron tomography on mitochondria, however, can be challenging. In this chapter, we describe the crucial steps from sample preparation to data acquisition enabling studies of mitochondrial translation in situ by cryoelectron tomography. We provide detailed protocols for yeast and human mitochondria preparations yielding a high concentration of intact mitochondrial vesicles on cryo-EM grids. In addition, we describe a workflow for particle identification and spatial mapping in context of the organelle., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)