Marburg virus (MARV) causes lethal hemorrhagic fever in humans, posing a threat to global health. We determined by cryogenic electron microscopy (cryo-EM) the MARV helical ribonucleoprotein (RNP) complex structure in single-layered conformation, which differs from the previously reported structure of a double-layered helix. Our findings illuminate novel RNP interactions and expand knowledge on MARV genome packaging and nucleocapsid assembly, both processes representing attractive targets for the development of antiviral therapeutics against MARV disease.

Most scientific facilities produce large amounts of heterogeneous data at a rapid pace. Managing users, instruments, reports and invoices presents additional challenges. To address these challenges, EMhub, a web platform designed to support the daily operations and record-keeping of a scientific facility, has been introduced. EMhub enables the easy management of user information, instruments, bookings and projects. The application was initially developed to meet the needs of a cryoEM facility, but its functionality and adaptability have proven to be broad enough to be extended to other data-generating centers. The expansion of EMHub is enabled by the modular nature of its core functionalities. The application allows external processes to be connected via a REST API, automating tasks such as folder creation, user and password generation, and the execution of real-time data-processing pipelines. EMhub has been used for several years at the Swedish National CryoEM Facility and has been installed in the CryoEM center at the Structural Biology Department at St. Jude Children’s Research Hospital. A fully automated single-particle pipeline has been implemented for on-the-fly data processing and analysis. At St. Jude, the X-Ray Crystallography Center and the Single-Molecule Imaging Center have already expanded the platform to support their operational and data-management workflows.

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.

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 angstrom resolution. SiaM comprises a transport domain and a scaffold domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na+ and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an 'elevator-with-an-operator' mechanism. Bacteria and archaea use tripartite ATP-independent periplasmic (TRAP) transporters to import essential nutrients. Davies et al. report a high resolution structure of a TRAP and show that it uses an 'elevator-with-an operator' mechanism.

Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle. Enteroviruses are non-enveloped positive-sense RNA viruses that modulate cytoplasmic membranes for replication. To enlighten how enteroviruses assemble around nascent RNA genomes and get package into autophagosomes for release, Dahmane et al. perform cryo-electron tomography of poliovirus-infected cells. They find assembly intermediates that are only present on the cytosolic side of the replication compartment and provide evidence that host factor VPS34 is involved in progression of assembly intermediates.

A semiartificial photosynthesis approach that utilizes enzymes for solar fuel production relies on efficient photosensitizers that should match the enzyme activity and enable long-term stability. Polymer dots (Pdots) are biocompatible photosensitizers that are stable at pH 7 and have a readily modifiable surface morphology. Therefore, Pdots can be considered potential photosensitizers to drive such enzyme-based systems for solar fuel formation. This work introduces and unveils in detail the interaction within the biohybrid assembly composed of binary Pdots and the HydA1 [FeFe]-hydrogenase from Chlamydomonas reinhardtii. The direct attachment of hydrogenase on the surface of toroid-shaped Pdots was confirmed by agarose gel electrophoresis, cryogenic transmission electron microscopy (Cryo-TEM), and cryogenic electron tomography (Cryo-ET). Ultrafast transient spectroscopic techniques were used to characterize photoinduced excitation and dissociation into charges within Pdots. The study reveals that implementation of a donor–acceptor architecture for heterojunction Pdots leads to efficient subpicosecond charge separation and thus enhances hydrogen evolution (88 460 μmol_H2·g_H2ase^–1·h^–1). Adsorption of [FeFe]-hydrogenase onto Pdots resulted in a stable biohybrid assembly, where hydrogen production persisted for days, reaching a TON of 37 500 ± 1290 in the presence of a redox mediator. This work represents an example of a homogeneous biohybrid system combining polymer nanoparticles and an enzyme. Detailed spectroscopic studies provide a mechanistic understanding of light harvesting, charge separation, and transport studied, which is essential for building semiartificial photosynthetic systems with efficiencies beyond natural and artificial systems. 

The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations. The phosphatidylinositol-3-phosphate (PI3P) is generated by the lipid kinase VPS34, in the context of VPS34 complex I on autophagosomes or complex II on endosomes. Biochemical and structural analyses provide insights into the mechanism of both VPS34 complexes recruitment to and activation on membranes by specific Rab GTPases.

Scipion is a modular image-processing framework that integrates several software packages under a unified interface while taking care of file formats and conversions. Here, new developments and capabilities of the Scipion plugin for the widely used RELION software package are presented and illustrated with an image-processing pipeline for published data. The user interfaces of Scipion and RELION are compared and the key differences are highlighted, allowing this manuscript to be used as a guide for both new and experienced users of this software. Different on-the-fly image-processing options are also discussed, demonstrating the flexibility of the Scipion framework.