With the increasing spread of infectious diseases worldwide, there is an urgent need for novel strategies to combat them. Cryogenic sample electron microscopy (cryo-EM) techniques, particularly electron tomography (cryo-ET), have revolutionized the field of infectious disease research by enabling multiscale observation of biological structures in a near-native state. This review highlights the recent advances in infectious disease research using cryo-ET and discusses the potential of this structural biology technique to help discover mechanisms of infection in native environments and guiding in the right direction for future drug discovery.

Cryo-EM is a powerful tool in structural biology, providing insights through techniques like single-particle analysis (SPA) and cryogenic electron tomography (cryo-ET). In thick specimens, challenges arise as an exponentially larger fraction of the transmitted electrons lose energy from inelastic scattering and can no longer be properly focused as a result of chromatic aberrations in the post-specimen optics. Rather than filtering out the inelastic scattering at the price of reducing potential signal, as is done in energy-filtered transmission electron microscopy (EFTEM), we show how a dose-efficient and unfiltered image can be rapidly obtained using tilt-corrected bright-field scanning-TEM (tcBF-STEM) data collected on a pixelated detector. Enhanced contrast and a 3-5x improvement in collection efficiency are observed for 2D images of intact bacterial cells and large organelles using tcBF-STEM compared to EFTEM for thicknesses beyond 500 nm. As a proof of concept for the technique’s performance in structural determination, we present an SPA map at a 7 Å nominal resolution for a highly symmetric virus-like particle (VLP) with 789 particles. These findings suggest applications for tcBF-STEM in cryo-EM of thicker cellular volumes where current approaches struggle. ### Competing Interest Statement The authors have declared no competing interest.

Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the destruction of invading RNA and DNA. Type III systems are among the most widespread in nature, and emerging interest in harnessing these systems for biotechnology applications highlights the need for detailed structural analyses of representatives from diverse organisms. We performed cryo-EM reconstructions of the Type III-A Cas10-Csm effector complex from S. epidermidis bound to an intact, cognate target RNA and identified two oligomeric states, a 276 kDa complex and a 318 kDa complex. 3.1 Å density for the well-ordered 276 kDa complex allowed construction of atomic models for the Csm2, Csm3, Csm4 and Csm5 subunits within the complex along with the crRNA and target RNA. We also collected small-angle X-ray scattering data which was consistent with the 276 kDa Cas10-Csm architecture we identified. Detailed comparisons between the S. epidermidis Cas10-Csm structure and the well-resolved bacterial (S. thermophilus) and archaeal (T. onnurineus) Cas10-Csm structures reveal differences in how the complexes interact with target RNA and crRNA which are likely to have functional ramifications. These structural comparisons shed light on the unique features of Type III-A systems from diverse organisms and will assist in improving biotechnologies derived from Type III-A effector complexes.

Cryo-electron microscopy (cryoEM) has become a popular method for determining high-resolution structures of biomolecules. However, data processing can be time-consuming, particularly for new researchers entering the field. To improve data quality and increase data collection efficiency, several software packages have been developed for on-the-fly data processing with various degrees of automation. These software packages allow researchers to perform tasks such as motion correction, CTF estimation, 2D classification, and 3D reconstruction in real-time, with minimal human input. On-the-fly data processing can not only improve data collection efficiency but also increase the productivity of instrumentation in high demand. However, the various software packages available differ in their performance, computational requirements, and levels of automation. In this review, we describe the minimal metrics used to assess data quality during data collection, outline the features of an ideal on-the-fly data processing software systems, and provide results from using three of these systems.

Molecular complexes at the endoplasmic reticulum and mitochondria contact sites (ERMCS) are not well understood in mammalian cells. The ERMCS are emerging as critical signaling platforms where unique biochemistry and physiology happens in cells. Furthermore, ERMCS have recently been shown to be dysregulated in neurons during early stages of neurodegenerative diseases such as Alzheimer's, Parkinson's, and fronto-temporal dementia FTD. The molecular tethers that bring these organelles together are largely unknown in mammalian cells but an ERMCS tethering protein (PDZD8) has recently been discovered.

Cryo-focused ion beam (FIB) milling of vitrified specimens is emerging as a powerful method for in situ specimen preparation. It allows for the preservation of native and near-native conditions in cells, and can reveal the molecular structure of protein complexes when combined with cryo-electron tomography (cryo-ET) and sub-tomogram averaging. Cryo-FIB milling is often performed on plunge-frozen specimens of limited thickness. However, this approach may have several disadvantages, including low throughput for cells that are small, or at low concentration, or poorly distributed across accessible areas of the grid, as well as for samples that may adopt a preferred orientation. Here, we present a detailed description of the "Waffle Method" protocol for vitrifying thick specimens followed by a semi-automated milling procedure using the Thermo Fisher Scientific (TFS) Aquilos 2 cryo-FIB/scanning electron microscope (SEM) instrument and AutoTEM Cryo software to produce cryo-lamellae. With this protocol, cryo-lamellae may be generated from specimens, such as microsporidia spores, yeast, bacteria, and mammalian cells, as well as purified proteins and protein complexes. An experienced lab can perform the entire protocol presented here within an 8-hour working day, resulting in two to three cryo-lamellae with target thicknesses of 100-200 nm and dimensions of approximately 12 μm width and 15-20 μm length. For cryo-FIB/SEMs with particularly low-contamination chambers, the protocol can be extended to overnight milling, resulting in up to 16 cryo-lamellae in 24 h. Graphical abstract.

In-situ cryo-electron tomography has emerged as an attractive method for determining high-resolution structures of macromolecules in their native state. While it is possible to achieve sub-nanometer resolution, it can prove difficult to precisely determine the location of the particle of interest in-situ amongst the wide range of cellular structures and particles present in a cell. To overcome this limitation, we have attached a gold fiducial in situ to a mitochondrial protein that allowed for improved targeting of the protein, while acting to facilitate better tilt-series alignments.

Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy chain (CHC), clathrin light chain (CLC), and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here, we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2 appendages crosslink adjacent CHC β-propellers and that the appendage densities are enriched in CCV hexagonal faces. We resolve how adaptor protein 2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.

Clathrin-mediated vesicle trafficking involves at least two dozen crucial and auxiliary components. These work together to encapsulate a variety of cargo into lipid vesicles that are surrounded by cage-like structures: so-called clathrin coated vesicles (CCVs). The cage component of CCVs is made of clathrin heavy chain (CHC) oligomerized into often highly symmetric geometries. Clathrin light chain (CLC) has been shown to be a negative regulator of cage assembly in physiological pH. CLC consists of a central heptad repeat which is also the major CHC-binding domain, and three interspaced C-terminal α-helical domains. Beneath the cages, adaptors bind clathrin, cargo, and phospholipids to coordinate the maturation of CCVs. Structural studies of clathrin-adaptor interactions have been limited to isolated peptide motifs of adaptors and the β-propeller domain of CHC, but studies of the association of the entire CCV complex with CHC, CLC, adaptors, membrane, and cargo have been limited. Understanding of the structural mechanisms by which the coat coordinates rapid and reliable assembly and disassembly, especially in synapses, remains incomplete. Here we shed new light on the mechanisms of CCV functions, performing subparticle single particle cryo-EM analysis on intact (not reconstituted) CCVs purified from cow brain. We have classified native brain CCVs into five geometries including a new low-symmetry (C2) cage structure not previously observed. By splitting the different cage geometries into different unique subparticles, we additionally resolved the coiled-coil part of CHC to 5 Angstroms, identified a novel set of interactions between the β-propeller of CHC and its ankle domain, resolved C-terminal regions of CLC, and characterized the distribution of adaptors and their interactions with CHC. These structures of intact CCVs provide a wealth of information about the mechanisms of CCV assembly.

In COPII mediated vesicle formation, Sec13/Sec31 heterotetramers play a role in organizing the membranes into a spherical vesicle. There they oligomerize into a cage that interacts with the other COPII proteins to direct vesicle formation and concentrate cargo into a bud. In this role they must be flexible to accommodate different sizes and shapes of cargo, but also have elements that provide rigidity to help deform the membrane. Here we characterize the influence the C-terminal disordered region of Sec31 has on cage flexibility and rigidity. After deleting this region (residues 820–1220), we characterized Sec13/Sec31ΔC heterotetramers biophysically and structurally through cryo-EM. Our results show that Sec13/31ΔC self-assembles into canonical cuboctahedral cages in vitro at buffer conditions similar to wild type. The distribution of cage sizes indicated that unlike the wild type, Sec13/31ΔC cages have a more homogeneous geometry. However, the structure of cuboctahedrons exhibited more conformational heterogeneity than wild type. Through localized reconstruction of cage vertices and molecular dynamics flexible fitting we found a new hinge for the flexing of Sec31 β-propeller domain and more flexibility of the previously known hinge. Together, these results show that the C-terminal region of Sec31 regulates the flexing of other domains such that flexibility and rigidity are not compromised during transport of large and/or asymmetric cargo.

Recently, localized reconstruction algorithms have been used to determine the structure of full length Sec13/31 COPII vertices to a resolution of 12 Angstroms. Inherent flexibility of COPII cages, which is vital to their biological function, has hindered high-resolution reconstructions by introducing conformational heterogeneity into datasets. Localized reconstruction deals with the conformational heterogeneity of whole COPII cage particles by extracting the more homogenous vertices and classifying them separately. Although this method has successfully increased resolution, achieving density maps with a high enough resolution to demonstrate secondary structures requires multiple approaches. We have hypothesized that the C-terminal unstructured domain of Sec13/31 could degrade the resolution of single particle reconstructions of the COPII cage by causing misalignment of projections. However, truncating those domains increases the flexibility of the cages, but leaves the COPII proteins' propensity to assemble into cages unchanged. Using localized reconstruction of the COPII cage vertices, we will show the range of structures that the truncated Sec13/31 edges can adopt. This will hopefully shed light on the structural basis of the increased flexibility of cages, and provide a way for us to circumvent it.