Observation of a single protein by ultrafast X-ray diffraction.

The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes. This was first demonstrated on biological samples a decade ago on the giant mimivirus. Since then, a large collaboration has been pushing the limit of the smallest sample that can be imaged. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.

Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells.

Here, we present a protocol for assessing virus-infected cells using electron cryo-tomography (cryoET). It includes the basic workflows of seeding cells, plunge-freezing, clipping, cryo-focused ion beam milling (cryoFIB-milling), and cryoET, as well as two optional modules: micropatterning and live-cell fluorescence microscopy. We use an A549 human cell line and the virus HAdV5-pIX-mcherry in this protocol, but the comprehensive workflow can be easily transferred to other cell types and different types of virus infection or treatment. For complete details on the use and execution of this protocol, please refer to Pfitzner et al. (2021).

Human Adenovirus Type 5 Infection Leads to Nuclear Envelope Destabilization and Membrane Permeability Independently of Adenovirus Death Protein.

The human adenovirus type 5 (HAdV5) infects epithelial cells of the upper and lower respiratory tract. The virus causes lysis of infected cells and thus enables spread of progeny virions to neighboring cells for the next round of infection. The mechanism of adenovirus virion egress across the nuclear barrier is not known. The human adenovirus death protein (ADP) facilitates the release of virions from infected cells and has been hypothesized to cause membrane damage. Here, we set out to answer whether ADP does indeed increase nuclear membrane damage. We analyzed the nuclear envelope morphology using a combination of fluorescence and state-of-the-art electron microscopy techniques, including serial block-face scanning electron microscopy and electron cryo-tomography of focused ion beam-milled cells. We report multiple destabilization phenotypes of the nuclear envelope in HAdV5 infection. These include reduction of lamin A/C at the nuclear envelope, large-scale membrane invaginations, alterations in double membrane separation distance and small-scale membrane protrusions. Additionally, we measured increased nuclear membrane permeability and detected nuclear envelope lesions under cryoconditions. Unexpectedly, and in contrast to previous hypotheses, ADP did not have an effect on lamin A/C reduction or nuclear permeability.

Structure of a robust bacterial protein cage and its application as a versatile biocatalytic platform through enzyme encapsulation.

Using a newly discovered encapsulin from Mycolicibacterium hassiacum, several biocatalysts were packaged in this robust protein cage. The encapsulin was found to be easy to produce as recombinant protein. Elucidation of its crystal structure revealed that it is a spherical protein cage of 60 protomers (diameter of 23 nm) with narrow pores. By developing an effective coexpression and isolation procedure, the effect of packaging a variety of biocatalysts could be evaluated. It was shown that encapsulation results in a significantly higher stability of the biocatalysts. Most of the targeted cofactor-containing biocatalysts remained active in the encapsulin. Due to the restricted diameters of the encapsulin pores (5-9 Å), the protein cage protects the encapsulated enzymes from bulky compounds. The work shows that encapsulins may be valuable tools to tune the properties of biocatalysts such as stability and substrate specificity.

Cellular Electron Cryo-Tomography to Study Virus-Host Interactions.

Viruses are obligatory intracellular parasites that reprogram host cells upon infection to produce viral progeny. Here, we review recent structural insights into virus-host interactions in bacteria, archaea, and eukaryotes unveiled by cellular electron cryo-tomography (cryoET). This advanced three-dimensional imaging technique of vitreous samples in near-native state has matured over the past two decades and proven powerful in revealing molecular mechanisms underlying viral replication. Initial studies were restricted to cell peripheries and typically focused on early infection steps, analyzing surface proteins and viral entry. Recent developments including cryo-thinning techniques, phase-plate imaging, and correlative approaches have been instrumental in also targeting rare events inside infected cells. When combined with advances in dedicated image analyses and processing methods, details of virus assembly and egress at (sub)nanometer resolution were uncovered. Altogether, we provide a historical and technical perspective and discuss future directions and impacts of cryoET for integrative structural cell biology analyses of viruses.

Fluorescent protein tagging of adenoviral proteins pV and pIX reveals 'late virion accumulation compartment'.

The human adenovirus type 5 (HAdV5) causes disease of the upper and lower respiratory tract. The early steps of HAdV5 entry up to genome replication in the host nucleus have been extensively studied. However, late stages of infection remain poorly understood. Here, we set out to elucidate the spatiotemporal orchestration of late adenovirus nuclear remodeling in living cells. We generated virus mutants expressing fluorescently tagged protein IX (pIX) and protein V (pV), a capsid and viral genome associated protein, respectively. We found that during progeny virion production both proteins localize to a membrane-less, nuclear compartment, which is highly impermeable such that in immunofluorescence microscopy antibodies can hardly penetrate it. We termed this compartment 'late virion accumulation compartment' (LVAC). Correlation between light- and electron microscopy revealed that the LVAC contains paracrystalline arrays of viral capsids that arrange tightly packed within a honeycomb-like organization of viral DNA. Live-cell microscopy as well as FRAP measurements showed that the LVAC is rigid and restricts diffusion of larger molecules, indicating that capsids are trapped inside.

A Technical Introduction to Transmission Electron Microscopy for Soft-Matter: Imaging, Possibilities, Choices, and Technical Developments.

With a significant role in material sciences, physics, (soft matter) chemistry, and biology, the transmission electron microscope is one of the most widely applied structural analysis tool to date. It has the power to visualize almost everything from the micrometer to the angstrom scale. Technical developments keep opening doors to new fields of research by improving aspects such as sample preservation, detector performance, computational power, and workflow automation. For more than half a century, and continuing into the future, electron microscopy has been, and is, a cornerstone methodology in science. Herein, the technical considerations of imaging with electrons in terms of optics, technology, samples and processing, and targeted soft materials are summarized. Furthermore, recent advances and their potential for application to soft matter chemistry are highlighted.

Complexome Profiling Reveals Association of PPR Proteins with Ribosomes in the Mitochondria of Plants.

Mitochondrial transcripts are subject to a wealth of processing mechanisms including cis- and trans-splicing events, as well as base modifications (RNA editing). Hundreds of proteins are required for these processes in plant mitochondria, many of which belong to the pentatricopeptide repeat (PPR) protein superfamily. The structure, localization, and function of these proteins is only poorly understood. Here we present evidence that several PPR proteins are bound to mitoribosomes in plants. A novel complexome profiling strategy in combination with chemical crosslinking has been employed to systematically define the protein constituents of the large and the small ribosomal subunits in the mitochondria of plants. We identified more than 80 ribosomal proteins, which include several PPR proteins and other non-conventional ribosomal proteins. These findings reveal a potential coupling of transcriptional and translational events in the mitochondria of plants. Furthermore, the data indicate an extremely high molecular mass of the "small" subunit, even exceeding that of the "large" subunit.

Solvent Mixing To Induce Molecular Motor Aggregation into Bowl-Shaped Particles: Underlying Mechanism, Particle Nature, and Application To Control Motor Behavior.

Control over dynamic functions in larger assemblies is key to many molecular systems, ranging from responsive materials to molecular machines. Here we report a molecular motor that forms bowl-shaped particles in water and how confinement of the molecular motor affects rotary motion. Studying the aggregation process in a broader context, we provide evidence that, in the case of bowl-shaped particles, the structures are not the product of self-assembly, but a direct result of the mixing a good solvent and a (partial) non-solvent and highly independent of the molecular design. Under the influence of the non-solvent, droplets are formed, of which the exterior is hardened due to the increase in the glass transition temperature by the external medium, while the interior of the droplets remains plasticized by the solvent, resulting in the formation of stable bowl-shaped particles with a fluid interior, a glass-like exterior, and a very specific shape: dense spheres with a hole in their side. Applying this to a bulky first-generation molecular motor allowed us to change its isomerization behavior. Furthermore, the motor shows in situ photo-switchable aggregation-induced emission. Strong confinement prohibits the thermal helix inversion step while altering the energy barriers that determine the rotary motion, such that it introduces a reverse trans- cis isomerization upon heating. These studies show a remarkable control of forward and backward rotary motion by simply changing solvent ratios and extent of confinement.

A general mechanism of ribosome dimerization revealed by single-particle cryo-electron microscopy.

Bacteria downregulate their ribosomal activity through dimerization of 70S ribosomes, yielding inactive 100S complexes. In Escherichia coli, dimerization is mediated by the hibernation promotion factor (HPF) and ribosome modulation factor. Here we report the cryo-electron microscopy study on 100S ribosomes from Lactococcus lactis and a dimerization mechanism involving a single protein: HPFlong. The N-terminal domain of HPFlong binds at the same site as HPF in Escherichia coli 100S ribosomes. Contrary to ribosome modulation factor, the C-terminal domain of HPFlong binds exactly at the dimer interface. Furthermore, ribosomes from Lactococcus lactis do not undergo conformational changes in the 30S head domains upon binding of HPFlong, and the Shine-Dalgarno sequence and mRNA entrance tunnel remain accessible. Ribosome activity is blocked by HPFlong due to the inhibition of mRNA recognition by the platform binding center. Phylogenetic analysis of HPF proteins suggests that HPFlong-mediated dimerization is a widespread mechanism of ribosome hibernation in bacteria.When bacteria enter the stationary growth phase, protein translation is suppressed via the dimerization of 70S ribosomes into inactive complexes. Here the authors provide a structural basis for how the dual domain hibernation promotion factor promotes ribosome dimerization and hibernation in bacteria.

Steering Self-Assembly of Amphiphilic Molecular Nanostructures via Halogen Exchange.

In the field of self-assembly, the quest for gaining control over the supramolecular architecture without affecting the functionality of the individual molecular building blocks is intrinsically challenging. By using a combination of synthetic chemistry, cryogenic transmission electron microscopy, optical absorption measurements, and exciton theory, we demonstrate that halogen exchange in carbocyanine dye molecules allows for fine-tuning the diameter of the self-assembled nanotubes formed by these molecules, while hardly affecting the molecular packing determined by hydrophobic/hydrophilic interactions. Our findings open a unique way to study size effects on the optical properties and exciton dynamics of self-assembled systems under well-controlled conditions.

Transmission Electron Microscopy as a Tool for the Characterization of Soft Materials: Application and Interpretation.

Transmission electron microscopy (TEM) provides direct structural information on nano-structured materials and is popular as a characterization tool in soft matter and supramolecular chemistry. However, technical aspects of sample preparation are overlooked and erroneous image interpretations are regularly encountered in the literature. There are three most commonly used TEM methods as we derived from literature: drying, staining and cryo-TEM, which are explained here with respect to their application, limitations and interpretation. Since soft matter chemistry relies on a lot of indirect evidence, the role of TEM for the correct evaluation of the nature of an assembly is very large. Mistakes in application and interpretation can therefore have enormous impact on the quality of present and future studies. We provide helpful background information of these three techniques, the information that can and cannot be derived from them and provide assistance in selecting the right technique for soft matter imaging. This essay warns against the use of drying and explains why. In general cryo-TEM is by far the best suited method and many mistakes and over-interpretations can be avoided by the use of this technique.

Enhanced retention of encapsulated ions in cross-linked polymersomes.

Polymer vesicles (polymersomes) composed of poly(butadiene-b-poly(ethylene oxide)) (PB-b-PEO) are known for their stability and limited permeability. However, when these vesicles are diluted, substances, such as ions, encapsulated in the aqueous cavity can be released due to vesicle disruption. In previous studies, we have shown that these vesicles can be loaded efficiently with sufficient quantities of radionuclides to allow application in radionuclide therapy and pharmacokinetics evaluation, provided that there is no loss of the encapsulated radionuclides when diluted in the bloodstream. In this paper, in order to stabilize the carriers, we propose to cross-link the hydrophobic part of the polymersome membrane and to investigate whether such cross-linking induced by γ radiation can enhance the retention of ions (radionuclides). Retention of ions encapsulated in the lumen in such cross-linked carriers has not been previously quantitatively evaluated, although it is of ultimate importance in any medical application. Here, we also investigate how cross-linking affects the transport of radionuclides (loading) through the membrane of the vesicles. The integrity of the vesicles as a function of the radiation dose is also investigated, including morphological changes. The results show that cross-linking hinders the transport of ions through the membrane, which also leads to higher retention of ions encapsulated prior to cross-linking in the vesicles. Electron micrographs show that the shape of the polymersomes is not greatly affected by γ radiation when left in the original solvent (phosphate buffered saline (PBS) or Milli-Q water), but when diluted in a good solvent for both blocks, i.e., tetrahydrofuran (THF), disintegration of the vesicles and the appearance of droplet-like structures is observed, which had not been reported previously. The results of the present study help to formulate polymersomes as carriers for radionuclide therapy, demonstrating a way to prevent in vivo release of radionuclides, caused by dilution-induced destabilization of the nanocarriers.

The 5 kDa protein NdhP is essential for stable NDH-1L assembly in Thermosynechococcus elongatus.

The cyanobacterial NADPH:plastoquinone oxidoreductase complex (NDH-1), that is related to Complex I of eubacteria and mitochondria, plays a pivotal role in respiration as well as in cyclic electron transfer (CET) around PSI and is involved in a unique carbon concentration mechanism (CCM). Despite many achievements in the past, the complex protein composition and the specific function of many subunits of the different NDH-1 species remain elusive. We have recently discovered in a NDH-1 preparation from Thermosynechococcus elongatus two novel single transmembrane peptides (NdhP, NdhQ) with molecular weights below 5 kDa. Here we show that NdhP is a unique component of the ∼ 450 kDa NDH-1L complex, that is involved in respiration and CET at high CO2 concentration, and not detectable in the NDH-1MS and NDH-1MS' complexes that play a role in carbon concentration. C-terminal fusion of NdhP with his-tagged superfolder GFP and the subsequent analysis of the purified complex by electron microscopy and single particle averaging revealed its localization in the NDH-1L specific distal unit of the NDH-1 complex, that is formed by the subunits NdhD1 and NdhF1. Moreover, NdhP is essential for NDH-1L formation, as this type of NDH-1 was not detectable in a ΔndhP::Km mutant.

Lactococcus lactis†YfiA is necessary and sufficient for ribosome dimerization.

Dimerization and inactivation of ribosomes in Escherichia coli is a two-step process that involves the binding of ribosome modulation factor (RMF) and hibernation promotion factor (HPF). Lactococcus lactis MG1363 expresses a protein, YfiA(L) (l) , which associates with ribosomes in the stationary phase of growth and is responsible for dimerization of ribosomes. We show that full-length YfiA(L) (l) is necessary and sufficient for ribosome dimerization in L. lactis but also functions heterologously in vitro with E. coli ribosomes. Deletion of the yfiA gene has no effect on the growth rate but diminishes the survival of L. lactis under energy-starving conditions. The N-terminal domain of YfiA(L) (l) is homologous to HPF from E. coli, whereas the C-terminal domain has no counterpart in E. coli. By assembling ribosome dimers in vitro, we could dissect the roles of the N- and C-terminal domains of YfiA(L) (l) . It is concluded that the dimerization and inactivation of ribosomes in L. lactis and E. coli differ in several cellular and molecular aspects. In addition, two-dimensional maps of dimeric ribosomes from L. lactis obtained by single particle electron microscopy show a marked structural difference in monomer association in comparison to the ribosome dimers in E. coli.