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Self-assembly of proteins holds great promise for the bottom-up design and production of synthetic biomaterials. In conventional approaches, designer proteins are pre-programmed with specific recognition sites that drive the association process towards a desired organized state. Although proven effective, this approach poses restrictions on the complexity and material properties of the end-state. An alternative, hierarchical approach that has found wide adoption for inorganic systems, relies on the production of crystalline nanoparticles that become the building blocks of a next-level assembly process driven by oriented attachment (OA). As it stands, OA has not yet been observed for protein systems. Here we employ cryo-transmission electron microscopy (cryoEM) in the high nucleation rate limit of protein crystals and map the self-assembly route at molecular resolution. We observe the initial formation of facetted nanocrystals that merge lattices by means of OA alignment well before contact is made, satisfying non-trivial symmetry rules in the process. As these nanocrystalline assemblies grow larger we witness imperfect docking events leading to oriented aggregation into mesocrystalline assemblies. These observations highlight the underappreciated role of the interaction between crystalline nuclei, and the impact of OA on the crystallization process of proteins. Past studies on protein nucleation have focused on the routes that molecules follow towards a crystalline cluster, while possible interactions that may occur between nuclei have not been investigated. Here, the authors show that in the high supersaturation limit such interactions dominate the nucleation process in the form of inter-nucleus docking driving by oriented attachment.

Polymer-grafted chromatography media, especially ion exchangers, are high performance materials for protein purification. However, due to the pore size limitation, conventional chromatography beads are usually not considered for the downstream processing of large biomolecules such as virus-like particles (VLPs). Contrariwise, since the outer surface of the chromatography beads provides satisfactory binding capacity for VLPs and impurities of smaller size can bind inside of the beads, conventional porous beads should be considered for VLP capture and purification. We used HIV-1 gag VLPs with a diameter of 100–200 nm as a model to demonstrate that polymer-grafted anion exchangers are suitable for the purification of bionanoparticles. The equilibrium binding capacity was 1 × 1013 part/mL resin. Moderate salt concentration up to 100 mM NaCl did not affect binding, allowing direct loading of cell culture supernatant onto the column for purification. Dynamic binding capacity at 10% breakthrough, when loading cell culture supernatant, was approximately 6 × 1011 part/mL column; only 1-log lower than for monoliths. Endonuclease treatment of the cell culture supernatant did not increase the dynamic binding capacity, suggesting that dsDNA does not compete for the binding sites of VLPs. Nevertheless, due to simultaneous elution of particles and dsDNA, endonuclease treatment is required to reduce dsDNA contamination in the product. Proteomic analysis revealed that HIV-1 gag VLPs contain different host cell proteins in their cargo. This cargo is composed of conserved proteins and other proteins that vary from one particle population to another, as well as from batch to batch. This process allowed the separation of different particle populations. HIV-1 gag VLPs were directly captured and purified from cell culture supernatant with a total particle recovery in the elution of about 35%. Columns packed with beads can be scaled to practically any dimension and therefore a tailored design of the process is possible.

Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323–785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements.

Recombinant production of IgM antibodies poses a special challenge due to the complex structure of the proteins and their not yet fully elucidated interactions with the immune effector proteins, especially the complement system. In this study, we present transient expression of IgM antibodies (IgM617, IgM012 and IgM012_GL) in HEK cells and compared it to the well-established stable expression system in CHO cells. The presented workflow investigates quality attributes including productivity, polymer distribution, glycosylation, antibody structure and activation of the classical complement pathway. The HEK293E transient expression system is able to generate comparable amounts and polymer distribution as IgM stably produced in CHO. Although the glycan profile generated by HEK293E cells contained a lower degree of sialylation and a higher portion of oligomannose structures, the potency to activate the complement cascade was maintained. Electron microscopy also confirmed the structural integrity of IgM pentamers produced in HEK293E cells, since the conventional star-shaped structure is observed. From our studies, we conclude that the transient expression system provides an attractive alternative for rapid, efficient and high-throughput production of complex IgM antibodies with slightly altered post-translational modifications, but comparable structure and function.

In the endoplasmic reticulum, calreticulin acts as a chaperone and a Ca(2+)-signalling protein. At the cell surface, it mediates numerous important biological effects. The crystal structure of the human calreticulin globular domain was solved at 1.55 Å resolution. Interactions of the flexible N-terminal extension with the edge of the lectin site are consistently observed, revealing a hitherto unidentified peptide-binding site. A calreticulin molecular zipper, observed in all crystal lattices, could further extend this site by creating a binding cavity lined by hydrophobic residues. These data thus provide a first structural insight into the lectin-independent binding properties of calreticulin and suggest new working hypotheses, including that of a multi-molecular mechanism.

Immunoglobulins type-M (IgMs) are one of the first antibody classes mobilized during immune responses against pathogens and tumor cells. Binding to specific target antigens enables the interaction with the C1 complex which strongly activates the classical complement pathway. This biological function is the basis for the huge therapeutic potential of IgMs. But, due to their high oligomeric complexity, in vitro production, biochemical characterization, and biophysical characterization are challenging. In this study, we present recombinant production of two IgM models (IgM617 and IgM012) in pentameric and hexameric states and the evaluation of their polymer distribution using different biophysical methods (analytical ultracentrifugation, size exclusion chromatography coupled to multi-angle laser light scattering, mass photometry, and transmission electron microscopy). Each IgM construct is defined by a specific expression and purification pattern with different sample quality. Nevertheless, both purified IgMs were able to activate complement in a C1q-dependent manner. More importantly, BioLayer Interferometry (BLI) was used for characterizing the kinetics of C1q binding to recombinant IgMs. We show that recombinant IgMs possess similar C1q-binding properties as IgMs purified from human plasma.

Measles is a highly contagious human disease. We used cryo–electron microscopy and single particle–based helical image analysis to determine the structure of the helical nucleocapsid formed by the folded domain of the measles virus nucleoprotein encapsidating an RNA at a resolution of 4.3 angstroms. The resulting pseudoatomic model of the measles virus nucleocapsid offers important insights into the mechanism of the helical polymerization of nucleocapsids of negative-strand RNA viruses, in particular via the exchange subdomains of the nucleoprotein. The structure reveals the mode of the nucleoprotein-RNA interaction and explains why each nucleoprotein of measles virus binds six nucleotides, whereas the respiratory syncytial virus nucleoprotein binds seven. It provides a rational basis for further analysis of measles virus replication and transcription, and reveals potential targets for drug design.

Although structures of vitrified supramolecular complexes have been determined at near-atomic resolution, elucidating in situ molecular structure in living cells remains a challenge. Here, we report a straightforward liquid cell technique, originally developed for real-time visualization of dynamics at a liquid-gas interface using transmission electron microscopy, to image wet biological samples. Due to the scattering effects from the liquid phase, the micrographs display an amplitude contrast comparable to that observed in negatively stained samples. We succeed in resolving subunits within the protein complex GroEL imaged in a buffer solution at room temperature. Additionally, we capture various stages of virus cell entry, a process for which only sparse structural data exists due to their transient nature. To scrutinize the morphological details further, we used individual particle electron tomography for 3D reconstruction of each virus. These findings showcase this approach potential as an efficient, cost-effective complement to other microscopy technique in addressing biological questions at the molecular level. Electron microscopy of native hydrated biological samples close to physiological temperature is challenging. Here, authors encapsulate proteins and cells as an efficient, cost-effective complement to other microscopy technique in addressing biological questions at the molecular level.

Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms. Only praziquantel is available for treating schistosomiasis, a disease affecting >200 million people. Here, the authors identify compounds active against schistosome infections meeting the criteria for lead progression indicated by WHO with better activity against juvenile worms than praziquantel.

Fluorescent silica nanoparticles are widely used for various applications from mechanical reinforcement to biology. In many cases, their surface has to be tailored. Herein fluorescent silica nanoparticles are synthesized by a reverse micro-emulsion process and functionalized by silane coupling agents owning amino and thiol groups. The functionalization is then characterized by macroscopic well-known methods (zeta potential, hydrophilic to hydrophobic ratio, etc.) and an original method based onto TEM observations of the contrast between the silica core and the metallic ions chelated by the functional groups grafted onto the surface is also introduced. This method reveals that the functionalization is effective and that it occurs by “nano domains.” It is therefore possible to characterize the functionalization by in situ observations. Finally, the characterized nanoparticles are incorporated into a PMMA thin film. The fluorescence of the nanoparticles allows the monitoring of the level of dispersion of the nanoparticles within the polymer and confirms all the other characterizations.