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.

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.

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: HPF^long. The N-terminal domain of HPF^long binds at the same site as HPF in Escherichia coli 100S ribosomes. Contrary to ribosome modulation factor, the C-terminal domain of HPF^long binds exactly at the dimer interface. Furthermore, ribosomes from Lactococcus lactis do not undergo conformational changes in the 30S head domains upon binding of HPF^long, and the Shine–Dalgarno sequence and mRNA entrance tunnel remain accessible. Ribosome activity is blocked by HPF^long due to the inhibition of mRNA recognition by the platform binding center. Phylogenetic analysis of HPF proteins suggests that HPF^long-mediated dimerization is a widespread mechanism of ribosome hibernation in bacteria.

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^Ll, which associates with ribosomes in the stationary phase of growth and is responsible for dimerization of ribosomes. We show that full‐length YfiA^Ll 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^Ll 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^Ll. 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.

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.

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. Graphical Abstract Highlights Plant mitoribosomes contain several pentatricopeptide repeat proteins. The small mitoribosomal subunit is of an exceptionally large size. Protein units not directly related to translation may be attached to plant mitoribosomes to confer additional functions to these molecular machines.