The future of integrated structural biology.

Instruct-ERIC, "the European Research Infrastructure Consortium for Structural biology research," is a pan-European distributed research infrastructure making high-end technologies and methods in structural biology available to users. Here, we describe the current state-of-the-art of integrated structural biology and discuss potential future scientific developments as an impulse for the scientific community, many of which are located in Europe and are associated with Instruct. We reflect on where to focus scientific and technological initiatives within the distributed Instruct research infrastructure. This review does not intend to make recommendations on funding requirements or initiatives directly, neither at the national nor the European level. However, it addresses future challenges and opportunities for the field, and foresees the need for a stronger coordination within the European and international research field of integrated structural biology to be able to respond timely to thematic topics that are often prioritized by calls for funding addressing societal needs.

Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.

A hallmark of neurodegenerative diseases is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription, and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.

Visualizing chaperonin function in situ by cryo-electron tomography.

Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding1-3. The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and the lid-shaped cofactor GroES at their apertures. In vitro analyses of the chaperonin reaction have shown that substrate protein folds, unimpaired by aggregation, while transiently encapsulated in the GroEL central cavity by GroES4-6. To determine the functional stoichiometry of GroEL, GroES and client protein in situ, here we visualized chaperonin complexes in their natural cellular environment using cryo-electron tomography. We find that, under various growth conditions, around 55-70% of GroEL binds GroES asymmetrically on one ring, with the remainder populating symmetrical complexes. Bound substrate protein is detected on the free ring of the asymmetrical complex, defining the substrate acceptor state. In situ analysis of GroEL-GroES chambers, validated by high-resolution structures obtained in vitro, showed the presence of encapsulated substrate protein in a folded state before release into the cytosol. Based on a comprehensive quantification and conformational analysis of chaperonin complexes, we propose a GroEL-GroES reaction cycle that consists of linked asymmetrical and symmetrical subreactions mediating protein folding. Our findings illuminate the native conformational and functional chaperonin cycle directly within cells.

Structure and topography of the synaptic V-ATPase-synaptophysin complex.

Synaptic vesicles are organelles with a precisely defined protein and lipid composition1,2, yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single-particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains3. The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the proton gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters4,5. Synaptophysin6 and its paralogues synaptoporin7 and synaptogyrin8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin-knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin-knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin.

Autophagy preferentially degrades non-fibrillar polyQ aggregates.

Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.

Combinatorial selective ER-phagy remodels the ER during neurogenesis.

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions, ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis, where a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodelling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of the ER and the role of individual ER-phagy receptors is limited. Here we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodelling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodelling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both the magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodelling and versatile genetic toolkit provide a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.

In situ structure of actin remodeling during glucose-stimulated insulin secretion using cryo-electron tomography.

Actin mediates insulin secretion in pancreatic ß-cells through remodeling. Hampered by limited resolution, previous studies have offered an ambiguous depiction as depolymerization and repolymerization. We report the in situ structure of actin remodeling in INS-1E ß-cells during glucose-stimulated insulin secretion at nanoscale resolution. After remodeling, the actin filament network at the cell periphery exhibits three marked differences: 12% of actin filaments reorient quasi-orthogonally to the ventral membrane; the filament network mainly remains as cell-stabilizing bundles but partially reconfigures into a less compact arrangement; actin filaments anchored to the ventral membrane reorganize from a "netlike" to a "blooming" architecture. Furthermore, the density of actin filaments and microtubules around insulin secretory granules decreases, while actin filaments and microtubules become more densely packed. The actin filament network after remodeling potentially precedes the transport and release of insulin secretory granules. These findings advance our understanding of actin remodeling and its role in glucose-stimulated insulin secretion.

Vimentin regulates nuclear segmentation in neutrophils.

Granulocytes are indispensable for various immune responses. Unlike other cell types in the body, the nuclei of granulocytes, particularly neutrophils, are heavily segmented into multiple lobes. Although this distinct morphological feature has long been observed, the underlying mechanism remains incompletely characterized. In this study, we utilize cryo-electron tomography to examine the nuclei of mouse neutrophils, revealing the cytoplasmic enrichment of intermediate filaments on the concave regions of the nuclear envelope. Aided by expression profiling and immuno-electron microscopy, we then elucidate that the intermediate-filament protein vimentin is responsible for such perinuclear structures. Of importance, exogenously expressed vimentin in nonimmune cells is sufficient to form cytoplasmic filaments wrapping on the concave nuclear surface. Moreover, genetic deletion of the protein causes a significant reduction of the number of nuclear lobes in neutrophils and eosinophils, mimicking the hematological condition of the Pelger-Huët anomaly. These results have uncovered a new component establishing the nuclear segmentation of granulocytes.

Combinatorial selective ER-phagy remodels the ER during neurogenesis.

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis: a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodeling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of ER and the role of individual ER-phagy receptors is limited. Here, we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodeling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodeling and versatile genetic toolkit provides a quantitative framework for understanding contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.

Munc13- and SNAP25-dependent molecular bridges play a key role in synaptic vesicle priming.

Synaptic vesicle tethering, priming, and neurotransmitter release require a coordinated action of multiple protein complexes. While physiological experiments, interaction data, and structural studies of purified systems were essential for our understanding of the function of the individual complexes involved, they cannot resolve how the actions of individual complexes integrate. We used cryo-electron tomography to simultaneously image multiple presynaptic protein complexes and lipids at molecular resolution in their native composition, conformation, and environment. Our detailed morphological characterization suggests that sequential synaptic vesicle states precede neurotransmitter release, where Munc13-comprising bridges localize vesicles <10 nanometers and soluble N-ethylmaleimide-sensitive factor attachment protein 25-comprising bridges <5 nanometers from the plasma membrane, the latter constituting a molecularly primed state. Munc13 activation supports the transition to the primed state via vesicle bridges to plasma membrane (tethers), while protein kinase C promotes the same transition by reducing vesicle interlinking. These findings exemplify a cellular function performed by an extended assembly comprising multiple molecularly diverse complexes.

In situ structure of actin remodeling during glucose-stimulated insulin secretion using cryo-electron tomography.

Actin mediates insulin secretion from the pancreatic ß-cell through a remodeling process. Previous studies have been hampered by limited resolution, providing an ambiguous depiction of actin remodeling as a process that begins with depolymerization into actin monomers, followed by repolymerization into actin filaments. Here, we report the in situ structure of actin remodeling in INS-1E ß-cells during glucose-stimulated insulin secretion at nanoscale resolution. We demonstrate that actin remodeling occurs at the cell periphery rather than in the cell interior. The actin filament network at the cell periphery exhibits three marked differences after remodeling compared to those under basal conditions. First, approximately 12%of actin filaments reorient, their angle changing from 0-45° to 45-90° relative to the plasma membrane. Second, the actin filament network remains predominantly as cell-stabilizing bundles but partially reconfigures into a less compact arrangement. Third, actin filaments anchored to the plasma membrane reorganize from a "netlike" to a "blooming" architecture, featuring radial projections emanating from their anchor points. Remodeling precedes the transport of insulin secretory granulesto the plasma membrane and their release from it. Furthermore, the density of actin filaments and microtubules around insulin secretory granules is lowered after remodeling compared to the basal conditions, as expected for the subsequent granule transport and release. Finally, actin filaments and microtubules are more densely packed than under basal conditions. These findings advance our structural and functional understanding of actin remodeling during glucose-stimulated insulin secretion in pancreatic ß-cells.

In situ snapshots along a mammalian selective autophagy pathway.

Selective macroautophagy (hereafter referred to as autophagy) describes a process in which cytosolic material is engulfed in a double membrane organelle called an autophagosome. Autophagosomes are carriers responsible for delivering their content to a lytic compartment for destruction. The cargo can be of diverse origin, ranging from macromolecular complexes to protein aggregates, organelles, and even invading pathogens. Each cargo is unique in composition and size, presenting different challenges to autophagosome biogenesis. Among the largest cargoes targeted by the autophagy machinery are intracellular bacteria, which can, in the case of Salmonella, range from 2 to 5 µm in length and 0.5 to 1.5 µm in width. How phagophores form and expand on such a large cargo remains mechanistically unclear. Here, we used HeLa cells infected with an auxotrophic Salmonella to study the process of phagophore biogenesis using in situ correlative cryo-ET. We show that host cells generate multiple phagophores at the site of damaged Salmonella-containing vacuoles (SCVs). The observed double membrane structures range from disk-shaped to expanded cup-shaped phagophores, which have a thin intermembrane lumen with a dilating rim region and expand using the SCV, the outer membrane of Salmonella, or existing phagophores as templates. Phagophore rims establish different forms of contact with the endoplasmic reticulum (ER) via structurally distinct molecular entities for membrane formation and expansion. Early omegasomes correlated with the marker Double-FYVE domain-Containing Protein 1 (DFCP1) are observed in close association with the ER without apparent membrane continuity. Our study provides insights into the formation of phagophores around one of the largest selective cargoes.

The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds in a cellular system.

Amyloid-like aggregates of the microtubule-associated protein Tau are associated with several neurodegenerative disorders including Alzheimer's disease. The existence of cellular machinery for the removal of such aggregates has remained unclear, as specialized disaggregase chaperones are thought to be absent in mammalian cells. Here we show in cell culture and in neurons that the hexameric ATPase valosin-containing protein (VCP) is recruited to ubiquitylated Tau fibrils, resulting in their efficient disaggregation. Aggregate clearance depends on the functional cooperation of VCP with heat shock 70 kDa protein (Hsp70) and the ubiquitin-proteasome machinery. While inhibition of VCP activity stabilizes large Tau aggregates, disaggregation by VCP generates seeding-active Tau species as byproduct. These findings identify VCP as a core component of the machinery for the removal of neurodegenerative disease aggregates and suggest that its activity can be associated with enhanced aggregate spreading in tauopathies.

A high-confidence Physcomitrium patens plasmodesmata proteome by iterative scoring and validation reveals diversification of cell wall proteins during evolution.

Plasmodesmata (PD) facilitate movement of molecules between plant cells. Regulation of this movement is still not understood. Plasmodesmata are hard to study, being deeply embedded within cell walls and incorporating several membrane types. Thus, structure and protein composition of PD remain enigmatic. Previous studies of PD protein composition identified protein lists with few validations, making functional conclusions difficult. We developed a PD scoring approach in iteration with large-scale systematic localization, defining a high-confidence PD proteome of Physcomitrium patens (HC300). HC300, together with bona fide PD proteins from literature, were placed in Pddb. About 65% of proteins in HC300 were not previously PD-localized. Callose-degrading glycolyl hydrolase family 17 (GHL17) is an abundant protein family with representatives across evolutionary scale. Among GHL17s, we exclusively found members of one phylogenetic clade with PD localization and orthologs occur only in species with developed PD. Phylogenetic comparison was expanded to xyloglucan endotransglucosylases/hydrolases and Exordium-like proteins, which also diversified into PD-localized and non-PD-localized members on distinct phylogenetic clades. Our high-confidence PD proteome HC300 provides insights into diversification of large protein families. Iterative and systematic large-scale localization across plant species strengthens the reliability of HC300 as basis for exploring structure, function, and evolution of this important organelle.

Ebola and Marburg virus VP35 coiled-coil validated as antiviral target by tripartite split-GFP complementation.

Ebola virus (EBOV) and Marburg virus (MARV) are highly pathogenic viruses in humans, against which approved antivirals are lacking. During EBOV and MARV infection, coiled-coil mediated oligomerization is essential for the virion protein 35 (VP35) polymerase co-factor function and type I interferon antagonism, making VP35 coiled-coil an elective drug target. We established a tripartite split-green fluorescent protein (GFP) fluorescence complementation (FC) system based on recombinant GFP-tagged EBOV and MARV VP35, which probes VP35 coiled-coil assembly by monitoring fluorescence on E. coli colonies, or in vitro in 96/384-multiwell. Oligomerization-defective VP35 mutants showed that correct coiled-coil knobs-into-holes pairing within VP35 oligomer is pre-requisite for GFP tags and GFP detector to reconstitute fluorescing full-length GFP. The method was validated by screening a small compound library, which identified Myricetin and 4,5,6,7-Tetrabromobenzotriazole as inhibitors of EBOV and MARV VP35 oligomerization-dependent FC with low-micromolar IC50 values. These findings substantiate the VP35 coiled-coil value as antiviral target.

Cryo-electron tomography: The power of seeing the whole picture.

Cryo-electron tomography allows to visualize the molecular architecture of pristinely preserved cellular landscapes in three dimensions. In favorable scenarios, macromolecular and supramolecular structures can be determined in situ with near-atomic resolution. The aim of visual proteomics is the parallel identification and annotation of all densities in the tomograms allowing for a comprehensive description of the molecular sociology of cells.

In situ structural analysis reveals membrane shape transitions during autophagosome formation.

Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. The analysis uncovers an unexpectedly thin intermembrane distance that is dilated at the phagophore rim. Mapping of individual autophagic structures onto a timeline based on geometric features reveals a dynamical change of membrane shape and curvature in growing phagophores. Moreover, our tomograms show the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles, such as the vacuole and the endoplasmic reticulum (ER). Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores toward closure without a templating cargo.