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Kinetochores are large structures composed of hundreds of proteins that assemble onto centromeric DNA to form a microtubule-binding site that is essential for proper chromosomal segregation. The structure of budding-yeast kinetochore particles is now studied by EM and electron tomography, revealing a large central hub surrounded by multiple globular domains, and multiple attachment sites for microtubules.

High-resolution data are crucial for accurate structural modeling. Here, authors enhance MicroED data quality using energy filtering, achieving sub-atomic resolution protein data and uncovering diffuse scattering, offering detailed insights into protein structure and function.

Embedded within the complexity of biological systems lies a formidable task: deciphering the intricate architecture of macromolecules. In this Viewpoint, a panel of experts discuss the key challenges and opportunities of macromolecular structure determination, highlighting the crucial synergy between empirical experimentation and artificial intelligence-based techniques in unravelling these complexities.

Through a MicroED structure of NaK ion channel, Shian Liu and Tamir Gonen identify a process of dilation coupled with Na⁺ movement, providing mechanistic insights into ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics.

Expanding the complexity of genetically encoded peptides is a long-standing challenge at the intersection of chemistry and biology. Now it has been shown that linear peptides with a reactive N-terminal β- or γ-keto amide can be synthesized ribosomally and elaborated to generate atropisomeric and/or macrocyclic peptides with embedded pharmacophores.

Human lens clarity and function depends on well-organized cell junctions. Here, the authors used MicroED to reveal the 3.5 Å structure of MP20, showing that MP20 tetramers form adhesive junctions essential for maintaining lens transparency

Virus-induced cellular condensates are poorly understood. Using cryoET and cryoEM, the authors visualized the 3D “molecular sociology” of host-virus interactions and choreographed mammalian reovirus assembly and replication within these condensates.

A computational method is reported that can be used to design protein nanomaterials in which two distinct subunits co-assemble into a specific architecture; five 24-subunit cage-like protein nanomaterials are designed, and experiments show that their structures are in close agreement with the computational design models.

Secretins are bacterial outer membrane proteins involved in different pathways for protein secretion or macromolecular complex assembly. Secretin can form a large oligomeric pore, whose opening needs to be carefully regulated. Now cryo-EM analysis of the Vibrio cholerae secretin GspD reveals a closed channel, with a constricted periplasmic vestibule, offering insight into the mechanism of GspD opening during protein secretion.

Lipid flipping across membrane leaflets are vital yet enigmatic biological processes. Here, five cryo-EM structures provide snapshots to delineate a mechanism of omega-3 fatty acid flipping across the blood brain barrier.

This article reports sub- and near-atomic structures of triclinic lysozyme and serine protease proteinase K, respectively, providing first demonstrations of ab initio phasing using electron counted MicroED data to solve macromolecular structures.

The crystal structure of SLC38A9 from Danio rerio in complex with arginine in the cytosol-open conformation reveals the mechanism of substrate binding.

Here, authors demonstrate a method for milling vitrified biological material. Using correlative fluorescence and electron microscopy images, MicroED data is collected for the adenosine receptor.

Cellular nitrite is rapidly removed from the cell to prevent formation of the cytotoxic nitric oxide; here the X-ray crystal structure of NarK, a bacterial nitrate/nitrite transport protein, is determined with and without substrate.

High-resolution, three-dimensional protein structures can be solved using MicroED, an electron diffraction method that uses three-dimensional microcrystals. An improved MicroED data collection approach described here increases data quality and resolution and extends its broad applicability.

Fragmentation of large, imperfect crystals into nanocrystals by sonication, vortexing, or vigorous pipetting facilitates atomic-resolution analysis by the cryo-EM method MicroED.

Glucose transporters are a medically important class of membrane proteins often deregulated in diseases such as Type 2 diabetes. Here, Wisedchaisri et al. report the crystal structure of XylE in an inward-facing open conformation to provide a general mechanism of substrate transport for the sugar porter family of proteins.

We present a protocol for preparing microcrystal samples for cryo-EM diffraction imaging, including room temperature solid-state small molecules and soluble and membrane protein crystals.

MicroED is a cryo-EM technique for collecting electron diffraction data from microcrystals and nanocrystals. This protocol from Gonen and colleagues describes how to prepare the protein crystal samples, how to set up the electron microscope for MicroED, and diffraction data collection.

NMR structural data and biophysical and biological experiments show that the antifungal compound amphotericin is toxic because it acts as a sterol sponge by interacting with ergosterol on the fungal membrane and extracting it from within the membrane to the surface of the membrane.

In patients with sporadic Alzheimer’s disease part of the Asp23 residues are isomerized to L-isoaspartate (L-isoAsp23). Here the authors present the MicroED structures of wild-type and L-isoAsp23 Aβ 20–34 amyloid fibrils that both form tightly packed cores and self-associate through two distinct interfaces with one of these interfaces being strengthened by the isoaspartyl modification.

The validation and analysis of X-ray crystallographic data is essential for reproducibility and the development of crystallographic methods. Here, the authors describe a repository for crystallographic datasets and demonstrate some of the ways it could serve the crystallographic community.

Rab small G proteins regulate membrane trafficking events by recruiting effectors that mediate vesicle tethering. In vitro studies now suggest that Vps21 and other endosomal Rabs in budding yeast can undergo GTP-regulated Rab-Rab interactions that drive tethering in the absence of effectors, implying that they have an intrinsic tethering activity that may function in concert with conventional effectors.

Microcrystal electron diffraction (MicroED) can determine the structure of proteins from crystals that are orders of magnitude smaller than those used by X-ray methods. Here, the application of MicroED to protein–ligand complexes is reviewed.

The computational design of an extremely stable icosahedral self-assembling protein nanocage is presented; the icosahedron should be useful for applications ranging from calibrating fluorescence microscopy to drug delivery.

A short segment of α-synuclein called NACore (residues 68–78) is responsible for the formation of amyloid aggregates responsible for cytotoxicity in Parkinson disease; here the nanocrystal structure of this invisible-to-optical-microscopy segment is determined using micro-electron diffraction, offering insight into its function and simultaneously demonstrating the first use of micro-electron diffraction to solve a previously unknown protein structure.

Calmodulin (CaM) regulates a variety of membrane channels in response to Ca2+, but the precise mechanisms are still unclear. Now a combination of single-particle EM, molecular dynamics simulations and functional assays is used to elucidate the structure of Ca2+–CaM bound to the full-length aquaporin AQP0, revealing a cytoplasmic gate that is closed upon CaM binding to control channel permeability in an allosteric manner.

Studies of the hepatitis C virus internal ribosome entry site (IRES) mechanism have focused on how IRES assembles an 80S ribosome at the start codon. Structural and functional analyses demonstrate that mutations of an IRES domain that docks in the 40S subunit's decoding groove cause conformational changes and that the mutated domain decreases IRES activity by inhibiting ribosome translocation and, thereby, translation elongation.

Structural analyses of fibrils formed by segments from human TDP-43 RRM2 reveal extensive packing and positional polymorphism.

The kinetochore is a large protein complex that assembles on centromeric DNA and captures microtubules to mediate chromosome separation. These authors report the first purification of functional kinetochores. They also show that kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules and that tension increases the lifetimes of the attachments directly. These results provide evidence that tension selectively stabilises kinetochore–microtubule interactions.

MicroED structure of a peptide from the β2–α2 loop of the bank vole prion protein reveals a protofibril stabilized by a dense network of hydrogen bonds.

This paper reviews the cryo-EM technique of microcrystal electron diffraction (MicroED), providing a broad overview of the technique, the unique structures determined, and the opportunities for future development.