Integrating cellular electron microscopy with multimodal data to explore biology across space and time.

Biology spans a continuum of length and time scales. Individual experimental methods only glimpse discrete pieces of this spectrum but can be combined to construct a more holistic view. In this Review, we detail the latest advancements in volume electron microscopy (vEM) and cryo-electron tomography (cryo-ET), which together can visualize biological complexity across scales from the organization of cells in large tissues to the molecular details inside native cellular environments. In addition, we discuss emerging methodologies for integrating three-dimensional electron microscopy (3DEM) imaging with multimodal data, including fluorescence microscopy, mass spectrometry, single-particle analysis, and AI-based structure prediction. This multifaceted approach fills gaps in the biological continuum, providing functional context, spatial organization, molecular identity, and native interactions. We conclude with a perspective on incorporating diverse data into computational simulations that further bridge and extend length scales while integrating the dimension of time.

Serial Lift-Out: sampling the molecular anatomy of whole organisms.

Cryo-focused ion beam milling of frozen-hydrated cells and subsequent cryo-electron tomography (cryo-ET) has enabled the structural elucidation of macromolecular complexes directly inside cells. Application of the technique to multicellular organisms and tissues, however, is still limited by sample preparation. While high-pressure freezing enables the vitrification of thicker samples, it prolongs subsequent preparation due to increased thinning times and the need for extraction procedures. Additionally, thinning removes large portions of the specimen, restricting the imageable volume to the thickness of the final lamella, typically <300 nm. Here we introduce Serial Lift-Out, an enhanced lift-out technique that increases throughput and obtainable contextual information by preparing multiple sections from single transfers. We apply Serial Lift-Out to Caenorhabditis elegans L1 larvae, yielding a cryo-ET dataset sampling the worm's anterior-posterior axis, and resolve its ribosome structure to 7 Å and a subregion of the 11-protofilament microtubule to 13 Å, illustrating how Serial Lift-Out enables the study of multicellular molecular anatomy.

Cryo-plasma FIB/SEM volume imaging of biological specimens.

Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of subcellular structures on the mesoscale (10 nm to 10 µm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents' hydration shells for in situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining, and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM. We evaluated the choice of plasma ion source and imaging regimes to produce high-quality SEM images of a range of different biological samples. Using an automated workflow we produced three-dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20-50 nm. Additionally, a tag-free localisation tool for regions of interest is needed to drive the application of in situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 µm) to produce lamellae for cryogenic electron tomography.

Allosteric control of Ubp6 and the proteasome via a bidirectional switch.

The proteasome recognizes ubiquitinated proteins and can also edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology. In yeast, editing is mediated by deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome through deubiquitination and a noncatalytic effect. Here, we report cryo-EM structures of the proteasome bound to Ubp6, based on which we identify mutants in Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations define a conserved region that we term the ILR element. The ILR is found within the BL1 loop, which obstructs the catalytic groove in free Ubp6. Rpt1-ILR interaction opens the groove by rearranging not only BL1 but also a previously undescribed network of three interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition are linked in that they are eliminated by the same mutations. Ubp6 and ubiquitin together drive proteasomes into a unique conformation associated with proteasome inhibition. Thus, a multicomponent allosteric switch exerts simultaneous control over both Ubp6 and the proteasome.

A modular platform for automated cryo-FIB workflows.

Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new-generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with three-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

In situ cryo-electron tomography reveals gradient organization of ribosome biogenesis in intact nucleoli.

Ribosomes comprise a large (LSU) and a small subunit (SSU) which are synthesized independently in the nucleolus before being exported into the cytoplasm, where they assemble into functional ribosomes. Individual maturation steps have been analyzed in detail using biochemical methods, light microscopy and conventional electron microscopy (EM). In recent years, single particle analysis (SPA) has yielded molecular resolution structures of several pre-ribosomal intermediates. It falls short, however, of revealing the spatiotemporal sequence of ribosome biogenesis in the cellular context. Here, we present our study on native nucleoli in Chlamydomonas reinhardtii, in which we follow the formation of LSU and SSU precursors by in situ cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). By combining both positional and molecular data, we reveal gradients of ribosome maturation within the granular component (GC), offering a new perspective on how the liquid-liquid-phase separation of the nucleolus supports ribosome biogenesis.

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity.

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.

Cryo-EM structure of the cetacean morbillivirus nucleoprotein-RNA complex.

Cetacean morbillivirus (CeMV) is an emerging and highly infectious paramyxovirus that causes outbreaks in cetaceans and occasionally in pinnipeds, representing a major threat to biodiversity and conservation of endangered marine mammal populations in both hemispheres. As for all non-segmented, negative-sense, single-stranded RNA (ssRNA) viruses, the morbilliviral genome is enwrapped by thousands of nucleoprotein (N) protomers. Each bound to six ribonucleotides, N protomers assemble to form a helical ribonucleoprotein (RNP) complex that serves as scaffold for nucleocapsid formation and as template for viral replication and transcription. While the molecular details on RNP complexes elucidated in human measles virus (MeV) served as paradigm model for these processes in all members of the Morbillivirus genus, no structural information has been obtained from other morbilliviruses, nor has any CeMV structure been solved so far. We report the structure of the CeMV RNP complex, reconstituted in vitro upon binding of recombinant CeMV N to poly-adenine ssRNA hexamers and solved to 4.0 Å resolution by cryo-electron microscopy. In spite of the amino acid sequence similarity and consequently similar folding of the N protomer, the CeMV RNP complex exhibits different helical parameters as compared to previously reported MeV orthologs. The CeMV structure reveals exclusive interactions leading to more extensive protomer-RNA and protomer-protomer interfaces. We identified twelve residues, among those varying between CeMV strains, as putatively important for the stabilization of the RNP complex, which highlights the need to study the potential of CeMV N mutations that modulate nucleocapsid assembly to also affect viral phenotype and host adaptation.

A streamlined workflow for automated cryo focused ion beam milling.

Cryo-electron tomography (cryo-ET) is an emerging technique to study the cellular architecture and the structure of proteins at high resolution in situ. Most biological specimens are too thick to be directly investigated and are therefore thinned by milling with a focused ion beam under cryogenic conditions (cryo-FIB). This procedure is prone to contaminations, which makes it a tedious process, often leading to suboptimal results. Here, we present new hardware that overcomes the current limitations. We developed a new glove box and a high vacuum cryo transfer system and installed a stage heater, a cryo-shield and a cryo-shutter in the FIB milling microscope. This reduces the ice contamination during the transfer and milling process and simplifies the handling of the sample. In addition, we tested a new software application that automates the key milling steps. Together, these improvements allow for high-quality, high-throughput cryo-FIB milling. This paves the way for new types of experiments, which have been previously considered infeasible.

High-resolution structure and biophysical characterization of the nucleocapsid phosphoprotein dimerization domain from the Covid-19 severe acute respiratory syndrome coronavirus 2.

Unprecedented by number of casualties and socio-economic burden occurring worldwide, the coronavirus disease 2019 (Covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the worst health crisis of this century. In order to develop adequate countermeasures against Covid-19, identification and structural characterization of suitable antiviral targets within the SARS-CoV-2 protein repertoire is urgently needed. The nucleocapsid phosphoprotein (N) is a multifunctional and highly immunogenic determinant of virulence and pathogenicity, whose main functions consist in oligomerizing and packaging the single-stranded RNA (ssRNA) viral genome. Here we report the structural and biophysical characterization of the SARS-CoV-2 N C-terminal domain (CTD), on which both N homo-oligomerization and ssRNA binding depend. Crystal structures solved at 1.44 Å and 1.36 Å resolution describe a rhombus-shape N CTD dimer, which stably exists in solution as validated by size-exclusion chromatography coupled to multi-angle light scattering and analytical ultracentrifugation. Differential scanning fluorimetry revealed moderate thermal stability and a tendency towards conformational change. Microscale thermophoresis demonstrated binding to a 7-bp SARS-CoV-2 genomic ssRNA fragment at micromolar affinity. Furthermore, a low-resolution preliminary model of the full-length SARS-CoV N in complex with ssRNA, obtained by cryo-electron microscopy, provides an initial understanding of self-associating and RNA binding functions exerted by the SARS-CoV-2 N.

Exploring the catalytic cascade of cembranoid biosynthesis by combination of genetic engineering and molecular simulations.

While chemical steps involved in bioactive cembranoid biosynthesis have been examined, the corresponding enzymatic mechanisms leading to their formation remain elusive. In the tobacco plant, Nicotiana tabacum, a putative cembratriene-ol synthase (CBTS) initiates the catalytic cascade that lead to the biosynthesis of cembratriene-4,6-diols, which displays antibacterial- and anti-proliferative activities. We report here on structural homology models, functional studies, and mechanistic explorations of this enzyme using a combination of biosynthetic and computational methods. This approach guided us to develop an efficient de novo production of five bioactive non- and monohydroxylated cembranoids. Our homology models in combination with quantum and classical simulations suggested putative principles of the CBTS catalytic cycle, and provided a possible rationale for the formation of premature olefinic side products. Moreover, the functional reconstruction of a N. tabacum-derived class II P450 with a cognate CPR, obtained by transcriptome mining provided for production of bioactive cembratriene-4,6-diols. Our combined findings provide mechanistic insights into cembranoid biosynthesis, and a basis for the sustainable industrial production of highly valuable bioactive cembranoids.

A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a.

Point-of-care testing (POCT) in low-resource settings requires tools that can operate independently of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably reaches a lower limit of detection (LOD) of ≈ 6.8 nM fluorescein, which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs with an LOD of 3.7 nM using a Cas13a-based fluorescence assay. Cas13a is an RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device may be used in educational settings, through providing low cost instrumentation for quantitative assays or as a platform to integrate hardware, software and biochemistry concepts in the future.