In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological breakthrough has provided insight into the structural and functional details of protein–lipid interactions. Most recently, advances in cryo-EM have demonstrated that SMA-extracted membrane proteins are a rich-source of structural data. For example, it has been possible to resolve the details of annular lipids and protein–protein interactions within complexes, the nature of lipids within central cavities and binding pockets, regions involved in stabilising multimers, details of terminal residues that would otherwise remain unresolved and the identification of physiologically relevant states. Functionally, SMA extraction has allowed the analysis of membrane proteins that are unstable in detergents, the characterization of an ultrafast component in the kinetics of electron transfer that was not possible in detergent-solubilised samples and quantitative, real-time measurement of binding assays with low concentrations of purified protein. While the use of SMA comes with limitations such as its sensitivity to low pH and divalent cations, its major advantage is maintenance of a protein's lipid bilayer. This has enabled researchers to view and assay proteins in an environment close to their native ones, leading to new structural and mechanistic insights.

Retromer (VPS26/VPS35/VPS29) is a highly conserved eukaryotic protein complex that localizes to endosomes to sort transmembrane protein cargoes into vesicles and elongated tubules. Retromer mediates retrieval pathways from endosomes to the trans -Golgi network in all eukaryotes and further facilitates recycling pathways to the plasma membrane in metazoans. In cells, retromer engages multiple partners to orchestrate the formation of tubulovesicular structures, including sorting nexin (SNX) proteins, cargo adaptors, GTPases, regulators, and actin remodeling proteins. Retromer-mediated pathways are especially important for sorting cargoes required for neuronal maintenance, which links retromer loss or mutations to multiple human brain diseases and disorders. Structural and biochemical studies have long contributed to the understanding of retromer biology, but recent advances in cryo-electron microscopy and cryo-electron tomography have further uncovered exciting new snapshots of reconstituted retromer structures. These new structures reveal retromer assembles into an arch-shaped scaffold and suggest the scaffold may be flexible and adaptable in cells. Interactions with cargo adaptors, particularly SNXs, likely orient the scaffold with respect to phosphatidylinositol-3-phosphate (PtdIns3 P )-enriched membranes. Pharmacological small molecule chaperones have further been shown to stabilize retromer in cultured cell and mouse models, but mechanisms by which these molecules bind remain unknown. This review will emphasize recent structural and biophysical advances in understanding retromer structure as the field moves towards a molecular view of retromer assembly and regulation on membranes.

This review discusses recent advances in single-particle cryo-EM and single-molecule approaches used to visualise eukaryotic DNA replication reactions reconstituted in vitro . We comment on the new challenges facing structural biologists, as they turn to describing the dynamic cascade of events that lead to replication origin activation and fork progression.

The progression of ESCRT (Endosomal Sorting Complexes Required for Transport) pathways, which mediate numerous cellular membrane fission events, is driven by the enzyme Vps4. Understanding of Vps4 mechanism is, therefore, of fundamental importance in its own right and, moreover, it is highly relevant to the understanding of many related AAA+ ATPases that function in multiple facets of cell biology. Vps4 unfolds its ESCRT-III protein substrates by translocating them through its central hexameric pore, thereby driving membrane fission and recycling of ESCRT-III subunits. This mini-review focuses on recent advances in Vps4 structure and mechanism, including ideas about how Vps4 translocates and unfolds ESCRT-III subunits. Related AAA+ ATPases that share structural features with Vps4 and likely utilize an equivalent mechanism are also discussed.

The spliceosome is a multi-subunit RNA–protein complex involved in the removal of non-coding segments (introns) from between the coding regions (exons) in precursors of messenger RNAs (pre-mRNAs). Intron removal proceeds via two transesterification reactions, occurring between conserved sequences at intron–exon junctions. A tightly regulated, hierarchical assembly with a multitude of structural and compositional rearrangements posed a great challenge for structural studies of the spliceosome. Over the years, X-ray crystallography dominated the field, providing valuable high-resolution structural information that was mostly limited to individual proteins and smaller sub-complexes. Recent developments in the field of cryo-electron microscopy allowed the visualisation of fully assembled yeast and human spliceosomes, providing unprecedented insights into substrate recognition, catalysis, and active site formation. This has advanced our mechanistic understanding of pre-mRNA splicing enormously.

Cryo-electron tomography (CET) is uniquely suited to obtain structural information from a wide range of biological scales, integrating and bridging knowledge from molecules to cells. In particular, CET can be used to visualise molecular structures in their native environment. Depending on the experiment, a varying degree of resolutions can be achieved, with the first near-atomic molecular structures becoming recently available. The power of CET has increased significantly in the last 5 years, in parallel with improvements in cryo-EM hardware and software that have also benefited single-particle reconstruction techniques. In this review, we cover the typical CET pipeline, starting from sample preparation, to data collection and processing, and highlight in particular the recent developments that support structural biology in situ . We provide some examples that highlight the importance of structure determination of molecules embedded within their native environment, and propose future directions to improve CET performance and accessibility.

Particles of cowpea mosaic virus (CPMV) have enjoyed considerable success as nanoparticles. The development of a system for producing empty virus-like particles (eVLPs) of the virus, which are non-infectious and have the potential to be loaded with heterologous material, has increased the number of possible applications for CPMV-based particles. However, for this potential to be realised, it was essential to demonstrate that eVLPs were accurate surrogates for natural virus particles, and this information was provided by high-resolution cryo-EM studies of eVLPs. This demonstration has enabled the approaches developed for the production of modified particles developed with natural CPMV particles to be applied to eVLPs. Furthermore, a combination of cryo-EM and mutagenic studies allowed the development of particles which are permeable but which could still assemble efficiently. These particles were shown to be loadable with cobalt, indicating that they can, indeed, be used as nano-containers.

The determination of detailed 3D structures of large and transient multicomponent complexes remains challenging. Here I describe the approaches that were used and developed by our laboratory to achieve structure solution of eukaryotic transcription complexes. I hope this collection serves as a resource for structural biologists seeking solutions for difficult structure determination projects.

High-resolution structures have given an extremely detailed view of aspects of ribosomes, including some near-functional states. Here, we review the importance of cryo-electron microscopy, among other techniques, in giving an understanding of the higher dynamics of the ribosome accompanying active recruitment of mRNA to the small subunit and translocation of tRNAs. Recent data show that careful use of a variety of different techniques is necessary for a proper understanding of the basis of function in systems such as the ribosome.

eEF2 (eukaryotic elongation factor 2) occupies an essential role in protein synthesis where it catalyses the translocation of the two tRNAs and the mRNA after peptidyl transfer on the 80 S ribosome. Recent crystal structures of eEF2 and the cryo-electron microscopy reconstruction of its 80 S complex now provide a substantial structural framework for dissecting the functional properties of this factor. The factor can be modified by either phosphorylation or ADP-ribosylation, which results in cessation of translation. We review the structural and functional properties of eEF2 with particular emphasis on the unique diphthamide residue, which is ADP-ribosylated by diphtheria toxin from Corynebacterium diphtheriae and exotoxin A from Pseudomonas aeruginosa .