Eukaryotic initiation factor 2 (eIF2) plays a key role in protein synthesis and in its regulation. The assembly of this heterotrimeric factor is facilitated by Cdc123, a member of the ATP grasp family that binds the γ subunit of eIF2. Notably, some mutations related to MEHMO syndrome, an X-linked intellectual disability, affect Cdc123-mediated eIF2 assembly. The mechanism of action of Cdc123 is unclear and structural information for the human protein is awaited. Here, the crystallographic structure of human Cdc123 (Hs-Cdc123) bound to domain 3 of human eIF2γ (Hs-eIF2γD3) was determined. The structure shows that the domain 3 of eIF2γ is bound to domain 1 of Cdc123. In addition, the long C-terminal region of Hs-Cdc123 provides a link between the ATP and Hs-eIF2γD3 binding sites. A thermal shift assay shows that ATP is tightly bound to Cdc123 whereas the affinity of ADP is much smaller. Yeast cell viability experiments, western blot analysis and two-hybrid assays show that ATP is important for the function of Hs-Cdc123 in eIF2 assembly. These data and recent findings allow us to propose a refined model to explain the mechanism of action of Cdc123 in eIF2 assembly.

Cryo-electron tomography and subtomogram averaging are rising and fast-evolving imaging techniques to study biological events, providing structural information at an unprecedented resolution while preserving spatial correlation in their native contexts. The latest technology and methodology development ranging from sample preparation to data collection and data processing, has enabled significant advancement in its applications to various biological systems. This review provides an overview of the current technology development enabling high-resolution structural study in situ, highlighting the use of a priori information of biological samples to assess the quality of subtomogram averaging pipeline. We exemplify the applications of this technique to understanding viruses and principles of macromolecule assembly using different biological systems, ranging from in vitro to in situ samples, which provide structural information at different resolutions and contexts.

Leucine Rich Repeat (LRR) domains, are present in hundreds of thousands of proteins across all kingdoms of life and are typically involved in protein-protein interactions and ligand recognition. LRR domains are classified into eight classes and when examined in three dimensions seven, of them form curved solenoid-like super-helices, also described as toruses, with a beta sheet on the concave (inside) and stacked alpha-helices on the convex (outside) of the torus. Here we present an overview of the least characterized 8th class of LRR proteins, the TpLRR-like LRRs, named after the Treponema pallidum protein Tp0225. Proteins from the TpLRR class differ from the proteins in all other known LRR classes by having a flipped curvature, with the beta sheet on the convex side of the torus and irregular secondary structure instead of helices on the opposite, now concave site. TpLRR proteins also present highly divergent sequence pattern of individual repeats and can associate with specific types of additional domains. Several of the characterized proteins from this class, specifically the BspA-like proteins, were found in human bacterial and protozoan pathogens, playing an important role in the interactions between the pathogens and the host immune system. In this paper we surveyed all existing experimental structures and selected AlphaFold models of the best-known proteins containing this class of LRR repeats, analyzing the relation between the pattern of conserved residues, specific structural features and functions of these proteins.

This paper introduces OpenFIBSEM, a universal API to control Focused Ion Beam Scanning Electron Microscopes (FIBSEM). OpenFIBSEM aims to improve the programmability and automation of electron microscopy workflows in structural biology research. The API is designed to be cross-platform, composable, and extendable: allowing users to use any portion of OpenFIBSEM to develop or integrate with other software tools. The package provides core functionality such as imaging, movement, milling, and manipulator control, as well as system calibration, alignment, and image analysis modules. Further, a library of reusable user interface components integrated with napari is provided, ensuring easy and efficient application development. OpenFIBSEM currently supports ThermoFisher and TESCAN hardware, with support for other manufacturers planned. To demonstrate the improved automation capabilities enabled by OpenFIBSEM, several example applications that are compatible with multiple hardware manufacturers are discussed. We argue that OpenFIBSEM provides the foundation for a cross-platform operating system and development ecosystem for FIBSEM systems. The API and applications are open-source and available on GitHub (https://github.com/DeMarcoLab/fibsem).

Advancements in the field of cryo-electron microscopy (cryo-EM) have greatly contributed to our current understanding of virus structures and life cycles. In this review, we discuss the application of single particle cryo-electron microscopy (EM) for the structure elucidation of small enveloped icosahedral viruses, namely, alpha- and flaviviruses. We focus on technical advances in cryo-EM data collection, image processing, three-dimensional reconstruction, and refinement strategies for obtaining high-resolution structures of these viruses. Each of these developments enabled new insights into the alpha- and flavivirus architecture, leading to a better understanding of their biology, pathogenesis, immune response, immunogen design, and therapeutic development.

Membrane proteins (MPs) are essential components of all biological membranes, contributing to key cellular functions that include signalling, molecular transport and energy metabolism. Consequently, MPs are important biomedical targets for therapeutics discovery. Despite hardware and software developments in cryo-electron microscopy, as well as MP sample preparation, MPs smaller than 100 kDa remain difficult to study structurally. Significant investment is required to overcome low levels of naturally abundant protein, MP hydrophobicity as well as conformational and compositional instability. Here we have reviewed the sample preparation approaches that have been taken to successfully express, purify and prepare small MPs for analysis by cryo-EM (those with a total solved molecular weight of under 100 kDa), as well as examining the differing approaches towards data processing and ultimately obtaining a structural solution. We highlight common challenges at each stage in the process as well as strategies that have been developed to overcome these issues. Finally, we discuss future directions and opportunities for the study of sub-100 kDa membrane proteins by cryo-EM.

Biomaterials for tissue regeneration must mimic the biophysical properties of the native physiological environment. A protein engineering approach allows the generation of protein hydrogels with specific and customised biophysical properties designed to suit a particular physiological environment. Herein, repetitive engineered proteins were successfully designed to form covalent molecular networks with defined physical characteristics able to sustain cell phenotype. Our hydrogel design was made possible by the incorporation of the SpyTag (ST) peptide and multiple repetitive units of the SpyCatcher (SC) protein that spontaneously formed covalent crosslinks upon mixing. Changing the ratios of the protein building blocks (ST:SC), allowed the viscoelastic properties and gelation speeds of the hydrogels to be altered and controlled. The physical properties of the hydrogels could readily be altered further to suit different environments by tuning the key features in the repetitive protein sequence. The resulting hydrogels were designed with a view to allow cell attachment and encapsulation of liver derived cells. Biocompatibility of the hydrogels was assayed using a HepG2 cell line constitutively expressing GFP. The cells remained viable and continued to express GFP whilst attached or encapsulated within the hydrogel. Our results demonstrate how this genetically encoded approach using repetitive proteins could be applied to bridge engineering biology with nanotechnology creating a level of biomaterial customisation previously inaccessible.

Proteins with two similar motifs in tandem are one of the most common cases of tandem repeat proteins. The question arises: why is the first emerged repeat frequently fixed in the process of evolution, despite the ample opportunities to continue its multiplication at the DNA level? To answer this question, we systematically analyzed the structure and function of these proteins. Our analysis showed that, in the vast majority of cases, the structural repetitive units have a two-fold (C2) internal symmetry. These closed structures provide an internal structural limitation for the subsequent growth of the repeat number. Frequently, the units "swap" their secondary structure elements with each other. Moreover, the duplicated domains, in contrast to other tandem repeat proteins, form binding sites for small molecules around the axis of C2 symmetry. Thus, the closure of the C2 structures and the emergence of new functional sites around the axis of C2 symmetry provide plausible explanations for why a repeat, once appeared, becomes fixed in the evolutionary process. We have placed these structures within the general structural classification of tandem repeat proteins, classifying them as either Class IV or V depending on the size of the repetitive unit.

Nucleocytoplasmatic large DNA viruses (NCLDVs or giant viruses) stand out because of their relatively large genomes encoding hundreds of proteins. These species give us an unprecedented opportunity to study the emergence and evolution of repeats in protein sequences. On the one hand, as viruses, these species have a restricted set of functions, which can help us better define the functional landscape of repeats. On the other hand, given the particular use of the genetic machinery of the host, it is worth asking whether this allows the variations of genetic material that lead to repeats in non-viral species. To support research in the characterization of repeat protein evolution and function, we present here an analysis focused on the repeat proteins of giant viruses, namely tandem repeats (TRs), short repeats (SRs), and homorepeats (polyX). Proteins with large and short repeats are not very frequent in non-eukaryotic organisms because of the difficulties that their folding may entail; however, their presence in giant viruses remarks their advantage for performance in the protein environment of the eukaryotic host. The heterogeneous content of these TRs, SRs and polyX in some viruses hints at diverse needs. Comparisons to homologs suggest that the mechanisms that generate these repeats are extensively used by some of these viruses, but also their capacity to adopt genes with repeats. Giant viruses could be very good models for the study of the emergence and evolution of protein repeats.

Alternative splicing of repeats in proteins provides a mechanism for rewiring and fine-tuning protein interaction networks. In this work, we developed a robust and versatile method, ASPRING, to identify alternatively spliced protein repeats from gene annotations. ASPRING leverages evolutionary meaningful alternative splicing-aware hierarchical graphs to provide maps between protein repeats sequences and 3D structures. We re-think the definition of repeats by explicitly accounting for transcript diversity across several genes/species. Using a stringent sequence-based similarity criterion, we detected over 5,000 evolutionary conserved repeats by screening virtually all human protein-coding genes and their orthologs across a dozen species. Through a joint analysis of their sequences and structures, we extracted specificity-determining sequence signatures and assessed their implication in experimentally resolved and modelled protein interactions. Our findings demonstrate the widespread alternative usage of protein repeats in modulating protein interactions and open avenues for targeting repeat-mediated interactions.

Membrane coat proteins are essential players in the eukaryotic endomembrane traffic system. Previous work identified proteins with the membrane-coat architecture in prokaryotes, specifically in the Planctomycetes, Verrucomicrobia and Chlamydiae (PVC) superphylum, bacteria that display the most developed prokaryotic endomembrane system. Hence, the membrane coat-like (MCL) proteins are predicted to play a central role in this system but their actual function is still unknown. In this work we strengthened previous structure predictions for these prokaryotic MCL proteins. We also detected new putative MCL proteins in the Planctomycete Gemmata obscuriglobus. Structural analysis of these revealed the presence of additional domains apart from the β-propeller and α-solenoid combination, characteristic of the membrane-coat architecture. Functions associated with these domains include some related to carbohydrate or membrane/lipid binding. Using homology-based methods, we found MCL proteins in other bacterial phyla, but the most abundant hits are still restricted to Planctomycetes and Verrucomicrobia. Detailed inspection of neighbouring genes of MCL in G. obscuriglobus supports the idea that the function of these proteins is related to membrane manipulation. No significant hits were found in Archaea, including Asgard archaea. More than 10 years after their original detection, PVC bacteria are still uniquely linked to eukaryotes through the structure of the MCL proteins sustaining their endomembrane system.

Structural biological hard tissues fulfill diverse tasks: protection, defence, locomotion, structural support, reinforcement, buoyancy. The cephalopod mollusk Spirula spirula has a planspiral, endogastrically coiled, chambered, endoskeleton consisting of the main elements: shell-wall, septum, adapical-ridge, siphuncular-tube. The cephalopod mollusk Sepia officinalis has an oval, flattened, layered-cellular endoskeleton, formed of the main elements: dorsal-shield, wall/pillar, septum, siphuncular-zone. Both endoskeletons are light-weight buoyancy devices that enable transit through marine environments: vertical (S. spirula), horizontal (S. officinalis). Each skeletal element of the phragmocones has a specific morphology, component structure and organization. The conjunction of the different structural and compositional characteristics renders the evolved nature of the endoskeletons and facilitates for Spirula frequent migration from deep to shallow water and for Sepia coverage over large horizontal distances, without damage of the buoyancy device. Based on Electron-Backscatter-Diffraction (EBSD) measurements and TEM, FE-SEM, laser-confocal-microscopy imaging we highlight for each skeletal element of the endoskeleton its specific mineral/biopolymer hybrid nature and constituent arrangement. We demonstrate that a variety of crystal morphologies and biopolymer assemblies are needed for enabling the endoskeleton to act as a buoyancy device. We show that all organic components of the endoskeletons have the structure of cholesteric-liquid-crystals and indicate which feature of the skeletal element yields the necessary mechanical property to enable the endoskeleton to fulfill its function. We juxtapose structural, microstructural, texture characteristics and benefits of coiled and planar endoskeletons and discuss how morphometry tunes structural biomaterial function. Both mollusks use their endoskeleton for buoyancy regulation, live and move, however, in distinct marine environments.

The different combinations of molecular dynamics simulations with coarse-grained representations have acquired considerable popularity among the scientific community. Especially in biocomputing, the significant speedup granted by simplified molecular models opened the possibility of increasing the diversity and complexity of macromolecular systems, providing realistic insights on large assemblies for more extended time windows. However, a holistic view of biological ensembles' structural and dynamic features requires a self-consistent force field, namely, a set of equations and parameters that describe the intra and intermolecular interactions among moieties of diverse chemical nature (i.e., nucleic and amino acids, lipids, solvent, ions, etc.). Nevertheless, examples of such force fields are scarce in the literature at the fully atomistic and coarse-grained levels. Moreover, the number of force fields capable of handling simultaneously different scales is restricted to a handful. Among those, the SIRAH force field, developed in our group, furnishes a set of topologies and tools that facilitate the setting up and running of molecular dynamics simulations at the coarse-grained and multiscale levels. SIRAH uses the same classical pairwise Hamiltonian function implemented in the most popular molecular dynamics software. In particular, it runs natively in AMBER and Gromacs engines, and porting it to other simulation packages is straightforward. This review describes the underlying philosophy behind the development of SIRAH over the years and across families of biological molecules, discussing current limitations and future implementations.

Cryo-electron tomography (cryoET) is a powerful technology that allows in-situ observation of the molecular structure of tissues and cells. Cryo-focused ion beam (cryoFIB) milling plays an important role in the preparation of high-quality thin lamellar samples for cryoET studies, thus, promoting the rapid development of cryoET in recent years. However, locating the regions of interest in a large cell or tissue during cryoFIB milling remains a major challenge limiting cryoET applications on arbitrary biological samples. Here, we report an on-the-fly localization method based on cellular secondary electron imaging (CSEI), which is derived from a basic imaging function of the cryoFIB instruments and enables high-contrast imaging of the cellular contents of frozen-hydrated biological samples. Moreover, CSEI does not require fluorescent labels and additional devices. The present study discusses the imaging principles and settings for optimizing CSEI. Tests on several commercially available cryoFIB instruments demonstrated that CSEI was feasible on mainstream instruments to observe all types of cellular contents and reliable under different milling conditions. We established a simple milling-localization workflow and tested it using the basal body of Chlamydomonas reinhardtii.

Water channels, which are small membrane proteins almost entirely buried in lipid membranes, are challenging research targets for single-particle cryo-electron microscopy (cryo-EM), a powerful technique routinely used to determine the structures of membrane proteins. Because the single-particle method enables structural analysis of a whole protein with flexible parts that interfere with crystallization, we have focused our efforts on analyzing water channel structures. Here, utilizing this system, we analyzed the structure of full-length aquaporin-2 (AQP2), a primary regulator of vasopressin-dependent reabsorption of water at the renal collecting ducts. The 2.9 Å resolution map revealed a cytoplasmic extension of the cryo-EM density that was presumed to be the highly flexible C-terminus at which the localization of AQP2 is regulated in the renal collecting duct cells. We also observed a continuous density along the common water pathway inside the channel pore and lipid-like molecules at the membrane interface. Observations of these constructions in the AQP2 structure analyzed without any fiducial markers (e.g., a rigidly bound antibody) indicate that single-particle cryo-EM will be useful for investigating water channels in native states as well as in complexes with chemical compounds.

Repetitivity and modularity of proteins are two related notions incorporated into multiple evolutionary concepts. We discuss whether they may also be essential for functional amyloids. Amyloids are proteins that create very regular and usually highly insoluble fibrils, which are often associated with neurodegeneration. However, recent discoveries showed that amyloid structure of a protein could also be beneficial and desired, e.g., to promote cell adhesion. Functional amyloids are proteins which differ in their characteristics from pathological amyloids, so that the fibril formation could be more under control of an organism. We propose that repeats in the sequence could regulate the aggregation propensity of these proteins. The inclusion of multiple symmetric interactions, due to the presence of the repeats, could be supporting and strengthening the desirable structural properties of functional amyloids. Our results show that tandem repeats in bacterial functional amyloids have a distinct characteristic. The pattern of repeats supports the appropriate level of fibril formation and better controllability of fibril stability. The repeats tend to be more imperfect, which attenuates excessive aggregation propensity. Their desired structure and function are also reinforced by their amino acid profile. Although in the study we focused on bacterial functional amyloids, due to their importance in biofilm formation, we propose that similar mechanisms could be employed in other functional amyloids which are designed by evolution to aggregate in a desirable manner, but not necessarily in pathological amyloids.

Structured tandem repeats proteins (STRPs) are a specific kind of tandem repeat proteins characterized by a modular and repetitive three-dimensional structure arrangement. The majority of STRPs adopt solenoid structures, but with the increasing availability of experimental structures and high-quality predicted structural models, more STRP folds can be characterized. Here, we describe "Box repeats", an overlooked STRP fold present in the DNA sliding clamp processivity factors, which has eluded classification although structural data has been available since the late 1990s. Each Box repeat is a β⍺βββ module of about 60 residues, which forms a class V "beads-on-a-string" type STRP. The number of repeats present in processivity factors is organism dependent. Monomers of PCNA proteins in both Archaea and Eukarya have 4 repeats, while the monomers of bacterial beta-sliding clamps have 6 repeats. This new repeat fold has been added to the RepeatsDB database, which now provides structural annotation for 66 Box repeat proteins belonging to different organisms, including viruses.

The HIV-1-encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with host proteins to support the virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. Here, we report on the Vpu oligomeric organization under membrane and aqueous conditions and provide insights into how the Vpu environment affects the oligomer formation. For these studies, we designed a maltose-binding protein (MBP)-Vpu chimera protein and produced it in E. coli in soluble form. We analyzed this protein using analytical size-exclusion chromatography (SEC), negative staining electron microscopy (nsEM), and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, we found that MBP-Vpu formed stable oligomers in solution, seemingly driven by Vpu transmembrane domain self-association. A coarse modeling of nsEM data as well as SEC and EPR data suggests that these oligomers most likely are pentamers, similar to what was reported regarding membrane-bound Vpu. We also noticed reduced MBP-Vpu oligomer stability upon reconstitution of the protein in β-DDM detergent and mixtures of lyso-PC/PG or DHPC/DHPG. In these cases, we observed greater oligomer heterogeneity, with MBP-Vpu oligomeric order generally lower than in solution; however, larger oligomers were also present. Notably, we found that in lyso-PC/PG, above a certain protein concentration, MBP-Vpu assembles into extended structures, which had not been reported for Vpu. Therefore, we captured various Vpu oligomeric forms, which can shed light on Vpu quaternary organization. Our findings could be useful in understanding Vpu organization and function in cellular membranes and could provide information regarding the biophysical properties of single-pass transmembrane proteins.

Coiled coils are a widespread and well understood protein fold. Their short and simple repeats underpin considerable structural and functional diversity. The vast majority of coiled coils consist of 7-residue (heptad) sequence repeats, but in essence most combinations of 3- and 4-residue segments, each starting with a residue of the hydrophobic core, are compatible with coiled-coil structure. The most frequent among these other repeat patterns are 11-residue (hendecad, 3 + 4 + 4) repeats. Hendecads are frequently found in low copy number, interspersed between heptads, but some proteins consist largely or entirely of hendecad repeats. Here we describe the first large-scale survey of these proteins in the proteome of life. For this, we scanned the protein sequence database for sequences with 11-residue periodicity that lacked β-strand prediction. We then clustered these by pairwise similarity to construct a map of potential hendecad coiled-coil families. Here we discuss these according to their structural properties, their potential cellular roles, and the evolutionary mechanisms shaping their diversity. We note in particular the continuous amplification of hendecads, both within existing proteins and de novo from previously non-coding sequence, as a powerful mechanism in the genesis of new coiled-coil forms.

Determination of sub-100 kDa (kDa) structures by cryo-electron microscopy (EM) is a longstanding but not straightforward goal. Here, we present a 2.9-Å cryo-EM structure of a 723-amino acid apo-form malate synthase G (MSG) from Escherichia coli. The cryo-EM structure of the 82-kDa MSG exhibits the same global folding as structures resolved by crystallography and nuclear magnetic resonance (NMR) spectroscopy, and the crystal and cryo-EM structures are indistinguishable. Analyses of MSG dynamics reveal consistent conformational flexibilities among the three experimental approaches, most notably that the α/β domain exhibits structural heterogeneity. We observed that sidechains of F453, L454, M629, and E630 residues involved in hosting the cofactor acetyl-CoA and substrate rotate differently between the cryo-EM apo-form and complex crystal structures. Our work demonstrates that the cryo-EM technique can be used to determine structures and conformational heterogeneity of sub-100 kDa biomolecules to a quality as high as that obtained from X-ray crystallography and NMR spectroscopy.

Single particle reconstruction (SPR) in cryoEM is an image processing task with an elaborate hierarchy that starts with many very noisy multi-frame images. Efficient representation of the intermediary image structures is critical for keeping the calculations manageable. One such intermediary structure is called a particle stack and contains cut-out images of particles in square boxes of predefined size. The micrograph that is the source of the boxed images is usually corrected for motion between frames prior to particle stack creation. However, the contrast transfer function (CTF) or its Fourier Transform point spread function (PSF) are not considered at this step. Historically, the particle stack was intended for large particles and for a tighter PSF, which is characteristic of lower resolution data. The field now performs analyses of smaller particles and to higher resolution, and these conditions result in a broader PSF that requires larger padding and slower calculations to integrate information for each particle. Consequently, the approach to handling structures such as the particle stack should be reexamined to optimize data processing. Here we propose to use as a source image for the particle stack a complex-valued image, in which CTF correction is implicitly applied as a real component of the image. We can achieve it by applying an initial CTF correction to the entire micrograph first and perform box cutouts as a subsequent step. The final CTF correction that we refine and apply later has a very narrow PSF, and so cutting out particles from micrographs that were approximately corrected for CTF does not require extended buffering, i.e. the boxes during the analysis only have to be large enough to encompass the particle. The Fourier Transform of an exit-wave reconstruction creates an image that has complex values. This is a complex value image considered in real space, opposed to standard SPR data processing where complex numbers appear only in Fourier space. This extension of the micrograph concept provides multiple advantages because the particle box size can be small and calculations crucial for high resolution reconstruction such as Ewald sphere correction, aberration refinement, and particle-specific defocus refinement can be performed on the small box data.

Particle localization (picking) in digital tomograms is a laborious and time-intensive step in cryogenic electron tomography (cryoET) analysis often requiring considerable user involvement, thus becoming a bottleneck for automated cryoET subtomogram averaging (STA) pipelines. In this paper, we introduce a deep learning framework called PickYOLO to tackle this problem. PickYOLO is a super-fast, universal particle detector based on the deep-learning real-time object recognition system YOLO (You Only Look Once), and tested on single particles, filamentous structures, and membrane-embedded particles. After training with the centre coordinates of a few hundred representative particles, the network automatically detects additional particles with high yield and reliability at a rate of 0.24-3.75 s per tomogram. PickYOLO can automatically detect number of particles comparable to those manually selected by experienced microscopists. This makes PickYOLO a valuable tool to substantially reduce the time and manual effort needed to analyse cryoET data for STA, greatly aiding in high-resolution cryoET structure determination.

The donut-shaped cucurbit[n]urils (Qn) are a class of rigid macrocyclic receptor with protein recognition capabilities. Qn encapsulation of amino acid side chains can enable protein assembly. Recently, cucurbit[7]uril (Q7) has been applied as a molecular glue for organizing protein building blocks into crystalline architectures. Q7 co-crystallization with dimethylated Ralstonia solanacearum lectin (RSL*) has yielded novel crystalline architectures. Co-crystallization of RSL* and Q7 yields either cage- or sheet-like architectures which may be modulated via protein engineering. However, questions remain as to the factors dictating the formation of one architecture over another (cage versus sheet). Here, we make use of an engineered RSL*-Q7 system which co-crystallizes as the cage or sheet assembly with easily-distinguished crystal morphologies. Using this model system, we probe how the crystallization conditions dictate which crystalline architecture is adopted. Protein-ligand ratios and the sodium concentration were identified as key determinants for the growth of the cage versus sheet assemblies.

The structural studies of macromolecules in their physiological context, particularly in tissue, is constrained by the bottleneck of sample preparation. In this study, we present a practical pipeline for preparing multicellular samples for cryo-electron tomography. The pipeline comprises sample isolation, vitrification, and lift-out-based lamella preparation using commercially available instruments. We demonstrate the efficacy of our pipeline by visualizing pancreatic β cells from mouse islets at the molecular level. This pipeline enables the determination of the properties of insulin crystals in situ for the first time, using unperturbed samples.

Peripheral membrane proteins are ubiquitous throughout cell biology and are required for a variety of cellular processes such as signal transduction, membrane trafficking, and autophagy. Transient binding to the membrane has a profound impact on protein function, serving to induce conformational changes and alter biochemical and biophysical parameters by increasing the local concentration of factors and restricting diffusion to two dimensions. Despite the centrality of the membrane in serving as a template for cell biology, there are few reported high-resolution structures of peripheral membrane proteins bound to the membrane. We analyzed the utility of lipid nanodiscs to serve as a template for cryo-EM analysis of peripheral membrane proteins. We tested a variety of nanodiscs and we report a 3.3 Å structure of the AP2 clathrin adaptor complex bound to a 17-nm nanodisc, with sufficient resolution to visualize a bound lipid head group. Our data demonstrate that lipid nanodiscs are amenable to high-resolution structure determination of peripheral membrane proteins and provide a framework for extending this analysis to other systems.

Synthetic ɑRep repeat proteins are engineered as Brick and Staple protein pairs that together self-assemble into helical filaments. In most cases, the filaments spontaneously form supercrystals. Here, we describe an expanded series of ɑRep Bricks designed to stabilize the interaction between consecutive Bricks, to control the length of the assembled multimers, or to alter the spatial distribution of the Staple on the filaments. The effects of these Brick modifications on the assembly, on the final filament structure and on the crystal symmetry are analyzed by biochemical methods, electron microscopy and small angle X-ray scattering. We further extend the concept of Brick/Staple protein origami by designing a new type of "Janus"-like Brick protein that is equally assembled by orthogonal staples binding its inner or outer surfaces and thus ending inside or outside the filaments. The relative roles of longitudinal and lateral associations in the assembly process are discussed. This set of results demonstrates important proofs-of-principle for engineering these remarkably versatile proteins toward nanometer-to-micron scale constructions.

NAD homeostasis in mammals requires the salvage of nicotinamide (Nam), which is cleaved from NAD+ by sirtuins, PARPs, and other NAD+-dependent signaling enzymes. Nam phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in vitamin B3 salvage, whereby Nam reacts with phosphoribosyl pyrophosphate (PRPP) to form nicotinamide mononucleotide. NAMPT has a high affinity towards Nam, which is further enhanced by autophosphorylation of His247. The mechanism of this enhancement has remained unknown. Here, we present high-resolution crystal structures and biochemical data that provide reasoning for the increased affinity of the phosphorylated NAMPT for its substrate. Structural and kinetic analyses suggest a mechanism that includes Mg2+ coordination by phospho-His247, such that PRPP is stabilized in a position highly favorable for catalysis. Under these conditions, nicotinic acid (NA) can serve as a substrate. Moreover, we demonstrate that a stretch of 10 amino acids, present only in NAMPTs from deuterostomes, facilitates conformational plasticity and stabilizes the chemically unstable phosphorylation of His247. Thereby the apparent substrate affinity is considerably enhanced compared to prokaryotic NAMPTs. Collectively, our study provides a structural basis for the important function of NAMPT to recycle Nam into NAD biosynthesis with high affinity.

The evolving SARS-CoV-2 Omicron strain has repeatedly caused widespread disease epidemics, and effective antibody drugs continue to be in short supply. Here, we identified a batch of nanobodies with high affinity for receptor binding domain (RBD) of SARS-CoV-2 spike protein, separated them into three classes using high performance liquid chromatography (HPLC), and then resolved the crystal structure of the ternary complexes of two non-competing nanobodies (NB1C6 and NB1B5) with RBD using X-ray crystallography. The structures showed that NB1B5 and NB1C6 bind to the left and right flank of the RBD, respectively, and that the binding epitopes are highly conserved cryptic sites in all SARS-CoV-2 mutant strains, as well as that NB1B5 can effectively block the ACE2. These two nanobodies were covalently linked into multivalent and bi-paratopic formats, and have a high affinity and neutralization potency for omicron, potentially inhibiting viral escape. The binding sites of these two nanobodies are relatively conserved, which help guide the structural design of antibodies targeting future variants of SARS-CoV-2 to combat COVID-19 epidemics and pandemics.

In cryo-transmission electron microscopy (cryo-TEM), sample thickness is one of the most important parameters that governs image quality. When combining cryo-TEM with other imaging methods, such as light microscopy, measuring and controlling the sample thickness to ensure suitability of samples becomes even more critical due to the low throughput of such correlated imaging experiments. Here, we present a method to assess the sample thickness using reflected light microscopy and machine learning that can be used prior to TEM imaging of a sample. The method makes use of the thin-film interference effect that is observed when imaging narrow-band LED light sources reflected by thin samples. By training a neural network to translate such reflection images into maps of the underlying sample thickness, we are able to accurately predict the thickness of cryo-TEM samples using a light microscope. We exemplify our approach using mammalian cells grown on TEM grids, and demonstrate that the thickness predictions are highly similar to the measured sample thickness. The open-source software described herein, including the neural network and algorithms to generate training datasets, is freely available at github.com/bionanopatterning/thicknessprediction. With the recent development of in situ cellular structural biology using cryo-TEM, there is a need for fast and accurate assessment of sample thickness prior to high-resolution imaging. We anticipate that our method will improve the throughput of this assessment by providing an alternative method to screening using cryo-TEM. Furthermore, we demonstrate that our method can be incorporated into correlative imaging workflows to locate intracellular proteins at sites ideal for high-resolution cryo-TEM imaging.

The remarkably complex skeletal systems of the sea stars (Echinodermata, Asteroidea), consisting of hundreds to thousands of individual elements (ossicles), have intrigued investigators for more than 150 years. While the general features and structural diversity of isolated asteroid ossicles have been well documented in the literature, the task of mapping the spatial organization of these constituent skeletal elements in a whole-animal context represents an incredibly laborious process, and as such, has remained largely unexplored. To address this unmet need, particularly in the context of understanding structure-function relationships in these complex skeletal systems, we present an integrated approach that combines micro-computed tomography, automated ossicle segmentation, data visualization tools, and the production of additively manufactured tangible models to reveal biologically relevant structural data that can be rapidly analyzed in an intuitive manner. In the present study, we demonstrate this high-throughput workflow by segmenting and analyzing entire skeletal systems of the giant knobby star, Pisaster giganteus, at four different stages of growth. The in-depth analysis, presented herein, provides a fundamental understanding of the three-dimensional skeletal architecture of the sea star body wall, the process of skeletal maturation during growth, and the relationship between skeletal organization and morphological characteristics of individual ossicles. The widespread implementation of this approach for investigating other species, subspecies, and growth series has the potential to fundamentally improve our understanding of asteroid skeletal architecture and biodiversity in relation to mobility, feeding habits, and environmental specialization in this fascinating group of echinoderms.

G protein-coupled receptors (GPCRs) are involved in a multitude of cellular signaling cascades and consequently are a prominent target for pharmaceutical drugs. In the past decades, a growing number of high-resolution structures of GPCRs has been solved, providing unprecedented insights into their mode of action. However, knowledge on the dynamical nature of GPCRs is equally important for a better functional understanding, which can be obtained by NMR spectroscopy. Here, we employed a combination of size exclusion chromatography, thermal stability measurements and 2D-NMR experiments for the NMR sample optimization of the stabilized neurotensin receptor type 1 (NTR1) variant HTGH4 bound to the agonist neurotensin. We identified the short-chain lipid di-heptanoyl-glycero-phosphocholine (DH7PC) as a promising membrane mimetic for high resolution NMR experiments and obtained a partial NMR backbone resonance assignment. However, internal membrane-incorporated parts of the protein were not visible due to lacking amide proton back-exchange. Nevertheless, NMR and hydrogen deuterium exchange (HDX) mass spectrometry experiments could be used to probe structural changes at the orthosteric ligand binding site in the agonist and antagonist bound states. To enhance amide proton exchange we partially unfolded HTGH4 and observed additional NMR signals in the transmembrane region. However, this procedure led to a higher sample heterogeneity, suggesting that other strategies need to be applied to obtain high-quality NMR spectra of the entire protein. In summary, the herein reported NMR characterization is an essential step toward a more complete resonance assignment of NTR1 and for probing its structural and dynamical features in different functional states.

Mitochondria are essential organelles that produce most of the energy via the oxidative phosphorylation (OXPHOS) system in all eukaryotic cells. Several essential subunits of the OXPHOS system are encoded by the mitochondrial genome (mtDNA) despite its small size. Defects in mtDNA maintenance and expression can lead to severe OXPHOS deficiencies and are amongst the most common causes of mitochondrial disease. The mtDNA is packaged as nucleoprotein structures, referred to as nucleoids. The conserved mitochondrial proteins, ARS-binding factor 2 (Abf2) in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and mitochondrial transcription factor A (TFAM) in mammals, are nucleoid associated proteins (NAPs) acting as condensing factors needed for packaging and maintenance of the mtDNA. Interestingly, gene knockout of Abf2 leads, in yeast, to the loss of mtDNA and respiratory growth, providing evidence for its crucial role. On a structural level, the condensing factors usually contain two DNA binding domains called high-mobility group boxes (HMG boxes). The co-operating mechanical activities of these domains are crucial in establishing the nucleoid architecture by bending the DNA strands. Here we used advanced solution NMR spectroscopy methods to characterize the dynamical properties of Abf2 together with its interaction with DNA. We find that the two HMG-domains react notably different to external cues like temperature and salt, indicating distinct functional properties. Biophysical characterizations show the pronounced difference of these domains upon DNA-binding, suggesting a refined picture of the Abf2 functional cycle.

The application of atomic force microscopy (AFM) for functional imaging and manipulating biomolecules at all levels of organization has enabled great progress in the structural biology field over the last decades, contributing to the discovery of novel structural entities of biological significance across many disciplines ranging from biochemistry, biomedicine and biophysics to molecular and cell biology, up to food systems and beyond. AFM has the capability to generate high-resolution topographic images spanning from the submolecular to the (sub)cellular range and can probe biochemical and biophysical sample properties in close to native conditions with excellent temporal resolution. Instrumental developments in the past decade enable dynamical structural and conformational studies of single biomolecules and new techniques for structural and chemical modification of the AFM probe have converted the cantilever into a versatile tool to study different biological phenomena, such as the mechanical stability of biomolecular complexes or the force induced dynamic changes of mechanically stressed proteins at the nanoscopic level. To improve the functionality of AFM and approach dynamic processes of complex biological systems ex vivo, AFM is combined with complementary microscopy, nanoscopy and spectroscopy tools. These multimethodological approaches provide unprecedented possibilities of probing physical, chemical and biological properties of complex cellular systems with high spatio-temporal resolution, leading to novel applications that correlate structural results with functional biochemical, biophysical, immunological, or genetic data of the system under study.

Cell recognition methods are in high demand in cell biology and medicine, and the method based on atomic force microscopy (AFM) shows a great value in application. The difference in mechanical properties or morphology of cells has been frequently used to detect whether cells are cancerous, but this detection method cannot be a general means for cancer cell detection, and the traditional artificial feature extraction method also has its limitations. In this work, we proposed an analytic method based on the physical properties of cells and deep learning method for recognizing cell types. The residual neural network used for recognition was modified by multi-scale convolutional fusion, attention mechanism and depthwise separable convolution, so as to optimize feature extraction and reduce operation costs. In the method, the collected cells were imaged by AFM, and the processed images were analyzed by the optimized convolutional neural network. The recognition results of two groups of cells (HL-7702 and SMMC-7721, SGC-7901 and GES-1) by this method show that the recognition rate of dataset with the combination of cell surface morphology, adhesion and Young's modulus is higher, and the recognition rate of the dataset with optimal resolution is higher. Our study indicated that the recognition of physical properties of cells using deep learning technology can serve as a universal and effective method for the automated analysis of cell information.

Spotted fever group Rickettsia undergo actin-based motility inside infected eukaryotic cells using Sca2 (surface cell antigen 2): an ∼ 1800 amino-acid monomeric autotransporter protein that is surface-attached to the bacterium and responsible for the assembly of long unbranched actin tails. Sca2 is the only known functional mimic of eukaryotic formins, yet it shares no sequence similarities to the latter. Using structural and biochemical approaches we have previously shown that Sca2 uses a novel actin assembly mechanism. The first ∼ 400 amino acids fold into helix-loop-helix repeats that form a crescent shape reminiscent of a formin FH2 monomer. Additionally, the N- and C- terminal halves of Sca2 display intramolecular interaction in an end-to-end manner and cooperate for actin assembly, mimicking a formin FH2 dimer. Towards a better structural understanding of this mechanism, we performed single-particle cryo-electron microscopy analysis of Sca2. While high-resolution structural details remain elusive, our model confirms the presence of a formin-like core: Sca2 indeed forms a doughnut shape, similar in diameter to a formin FH2 dimer and can accommodate two actin subunits. Extra electron density, thought to be contributed by the C-terminal repeat domain (CRD), covering one side is also observed. This structural analysis allows us to propose an updated model where nucleation proceeds by encircling two actin subunits, and elongation proceeds either by a formin-like mechanism that necessitates conformational changes in the observed Sca2 model, or via an insertional mechanism akin to that observed in the ParMRC system.

Molluscs rapidly repair the damaged shells to prevent further injury, which is vital for their survival after physical or biological aggression. However, it remains unclear how this process is precisely controlled. In this study, we applied scanning electronic microscope and histochemical analysis to examine the detailed shell regeneration process in the pearl oyster Pinctada fucata. It was found that the shell damage caused the mantle tissue to retract, which resulted in relocation of the partitioned mantle zones with respect to their correspondingly secreting shell layers. As a result, the relocated mantle tissue dramatically altered the shell morphology by initiating de novo precipitation of prismatic layers on the former nacreous layers, leading to the formation of sandwich-like "prism-nacre-prism-nacre" structure. Real-time PCR revealed the up-regulation of the shell matrix protein genes, which was confirmed by the thermal gravimetric analysis of the newly formed shell. The increased matrix secretion might have led to the change of CaCO3 precipitation dynamics which altered the mineral morphology and promoted shell formation. Taken together, our study revealed the close relationship between the physiological activities of the mantle tissue and the morphological change of the regenerated shells.

Repeat proteins are common in all domains of life and exhibit a wide range of functions. One class of repeat protein contains solenoid folds where the repeating unit consists of β-strands separated by tight turns. β-solenoids have distinguishing structural features such as handedness, twist, oligomerisation state, coil shape and size which give rise to their diversity. Characterised β-solenoid repeat proteins are known to form regions in bacterial and viral virulence factors, antifreeze proteins and functional amyloids. For many of these proteins, the experimental structure has not been solved, as they are difficult to crystallise or model. Here we use various deep learning-based structure-modelling methods to discover novel predicted β-solenoids, perform structural database searches to mine further structural neighbours and relate their predicted structure to possible functions. We find both eukaryotic and prokaryotic adhesins, confirming a known functional linkage between adhesin function and the β-solenoid fold. We further identify exceptionally long, flat β-solenoid folds as possible structures of mucin tandem repeat regions and unprecedentedly small β-solenoid structures. Additionally, we characterise a novel β-solenoid coil shape, the FapC Greek key β-solenoid as well as plausible complexes between it and other proteins involved in Pseudomonas functional amyloid fibres.

The cytoplasmatic biosynthesis of the stem peptide from the peptidoglycan in bacteria involves six steps, which have the role of three ATP-dependent Mur ligases that incorporate three consecutive amino acids to a substrate precursor. MurE is the last Mur ligase to incorporate a free amino acid. Although the structure of MurE from Mycobacterium tuberculosis (MtbMurE) was determined at 3.0 Å, the binding mode of meso-Diaminopimelate (m-DAP) and the effect of substrate absence is unknown. Herein, we show the structure of MurE from M. thermoresistibile (MthMurE) in complex with ADP and m-DAP at 1.4 Å resolution. The analysis of the structure indicates key conformational changes that the substrate UDP-MurNAc-L-Ala-D-Glu (UAG) and the free amino acid m-DAP cause on the MthMurE conformation. We observed several movements of domains or loop regions that displace their position in order to perform enzymatic catalysis. Since MthMurE has a high similarity to MtbMurE, this enzyme could also guide strategies for structure-based antimicrobial discovery to fight against tuberculosis or other mycobacterial infections.

While recent advances in cryo-EM, coupled with single particle analysis, have the potential to allow structure determination in a near-native state from vanishingly few individual particles, this vision has yet to be realised in practise. Requirements for particle numbers that currently far exceed the theoretical lower limits, challenges with the practicalities of achieving high concentrations for difficult-to-produce samples, and inadequate sample-dependent imaging conditions, all result in significant bottlenecks preventing routine structure determination using cryo-EM. Therefore, considerable efforts are being made to circumvent these bottlenecks by developing affinity purification of samples on-grid; at once obviating the need to produce large amounts of protein, as well as more directly controlling the variable, and sample-dependent, process of grid preparation. In this proof-of-concept study, we demonstrate a further practical step towards this paradigm, developing a 3D-printable flow-cell device to allow on-grid affinity purification from raw inputs such as whole cell lysates, using graphene oxide-based affinity grids. Our flow-cell device can be interfaced directly with routinely-used laboratory equipment such as liquid chromatographs, or peristaltic pumps, fitted with standard chromatographic (1/16") connectors, and can be used to allow binding of samples to affinity grids in a controlled environment prior to the extensive washing required to remove impurities. Furthermore, by designing a device which can be 3D printed and coupled to routinely used laboratory equipment, we hope to increase the accessibility of the techniques presented herein to researchers working towards single-particle macromolecular structures.

Of the two putative amino acid binding periplasmic receptors of ABC transporter family in Candidatus Liberibacter asiaticus (CLas), cystine binding receptor (CLasTcyA) has been shown to mainly express in phloem of citrus plant and is a target for inhibitor development. The crystal structure of CLasTcyA in complex with substrates has been reported earlier. The present work reports the identification and evaluation of potential candidates for their inhibitory potential against CLasTcyA. Among many compounds, selected through virtual screening, and MD simulation, pimozide, clidinium, sulfasalazine and folic acid showed significantly higher affinities and stability in complex with CLasTcyA. The SPR studies with CLasTcyA revealed significantly higher binding affinities for pimozide and clidinium (Kd, 2.73 nM and 70 nM, respectively) as compared to cystine (Kd, 1.26 µM). The higher binding affinities could be attributed to significantly increased number of interactions in the binding pocket as evident from the crystal structures of CLasTcyA in complex with pimozide and clidinium as compared to cystine. The CLasTcyA possess relatively large binding pocket where bulkier inhibitors fit quite well. In planta studies, carried out to assess the effect of inhibitors on HLB infected Mosambi plants, showed significant reduction in CLas titre in plants treated with inhibitors as compared to control plants. The results showed that pimozide exhibited higher efficiency as compared to clidinium in reducing CLas titre in treated plants. Our results showed that the inhibitor development against critical proteins like CLasTcyA can be an important strategy in management of HLB.

Single particle cryo-electron microscopy has become a critical tool in structural biology over the last decade, able to achieve atomic scale resolution in three dimensional models from hundreds of thousands of (noisy) two-dimensional projection views of particles frozen at unknown orientations. This is accomplished by using a suite of software tools to (i) identify particles in large micrographs, (ii) obtain low-resolution reconstructions, (iii) refine those low-resolution structures, and (iv) finally match the obtained electron scattering density to the constituent atoms that make up the macromolecule or macromolecular complex of interest. Here, we focus on the second stage of the reconstruction pipeline: obtaining a low resolution model from picked particle images. Our goal is to create an algorithm that is capable of ab initio reconstruction from small data sets (on the order of a few thousand selected particles). More precisely, we propose an algorithm that is robust, automatic, and fast enough that it can potentially be used to assist in the assessment of particle quality as the data is being generated during the microscopy experiment.

Corbicula clam shells consist of thin periostracum and calcareous layers made of calcium carbonate (CaCO3). Depending on habitat conditions, the shell exhibits various colorations, such as yellow, brown, and black. The chemical state of the periostracum of the Corbicula species in Lake Biwa was studied by X-ray absorption fine structure (XAFS) and Raman scattering spectroscopies. Fe K-edge X-ray absorption near edge structure (XANES) revealed that the Fe3+ intensity increases as the color of the shell changes from yellow to black. Raman spectra suggested that quinone-based polymers cover the yellow shell, and the black shell is further covered by dihydroxyphenylalanine (DOPA) rings of amino acid derivatives. From Fe K-edge extended X-ray absorption fine structure (EXAFS), it was found that Fe3+ in the periostracum was surrounded by five to six oxygen atoms with an average Fe-O ligand distance of 2.0 Å. Accordingly, a tris-DOPA-Fe3+ complex is formed, which is responsible for the periostracum's black color.

Cortisol is a steroid hormone that is produced by the adrenal gland. It is a primary stress hormone that increases glucose levels in the blood stream. High concentrations of cortisol in the body can be used as a biomarker for acute and chronic stress and related mental and physiological disorders. Therefore, the accurate quantification of cortisol levels in body fluids is essential for clinical diagnosis. In this article, we describe the isolation of recombinant anti-cortisol antibodies with high affinity for cortisol and discover their cross-reactivity with other glucocorticoids. To describe the cortisol binding site and elucidate the structural basis for the binding specificity, the high-resolution crystal structures of the anti-cortisol (17) Fab fragment in the absence of glucocorticoid (2.00 Å) and the presence of cortisol (2.26 Å), corticosterone (1.86 Å), cortisone (1.85 Å) and prednisolone (2.00 Å) were determined. To our knowledge, this is the first determined crystal structure of a cortisol-specific antibody. The recognition of cortisol is driven by hydrophobic interactions and hydrogen bonding at the protein-ligand interface coupled with a conformational transition. Comparison of ligand-free and ligand-bound structures showed that the side chains of residues Tyr58-H and Arg56-H can undergo local conformational changes at the binding site, most likely prior to the binding event via a conformational selection mechanism. Compared to other anti-steroid antibody-antigen complexes, (17) Fab possesses a structurally unique steroid binding site, as the H3 loop from the CDR area has only a minor contribution, but framework residues have a prominent contribution to hapten binding.

Elastic proteins and derived biomaterials contain numerous tandemly repeated peptides along their sequences, ranging from a few copies to hundreds. These repetitions are responsible for their biochemical, biological and biomechanical properties. These sequences are considered to be intrinsically disordered, and the variations in their behavior are actually mainly due to their high flexibility and lack of stable secondary structures originating from their unique amino acid sequences. Consequently, the simulation of elastic proteins and large elastomeric biomaterials using classical molecular dynamics is an important challenge. Here, we propose a novel approach that allows the application of the DURABIN protocol to repeated elastin-like peptides (r-ELPs) in a simple way. Four large r-ELPs were studied to evaluate our method, which was developed for simulating extracellular matrix proteins at the mesoscopic scale. After structure clustering applied on molecular dynamic trajectories of constitutive peptides (5-mers and 6-mers), the main conformations were used as starting points to define the corresponding primitives, further used as rigid body fragments in our program. Contributions derived from electrostatic and molecular hydrophobicity potentials were tested to evaluate their influence on the interactions during simple mesoscopic simulations. The CHLORAINE approach, despite the thinner granularity due to the size of the patterns used, was included in the DURABIN protocol and emerges as a promising way to simulate elastic macromolecular systems.

The gypsum habitats of Iran are significant reserves of biodiversity containing endemic and rare species. Despite the limited understanding of its characteristics and habitat, it has become essential to study the endemic species of the Semnan gypsic soil. Fresh samples of studied species including Acantholimon cymosum, Astragalus fridae, Astragalus semnanensis, Euphorbia gypsicola, Gypsophila mucronofolia, Moltkia gypsaceae and Nepeta eremokosmus were collected in the wild during the growth season. Leaf surface and leaf cross-sections were considered. The longest hair length was related to A. fridae, A. semnanensis and M. gypsaceae species. The shorter hairs belong to the species A. cymosum, G. mucronofolia and E. gypsicola. Crystals called cystolites were seen in the epidermal cell wall of A. semnanensis leaves. The anatomical characteristics of these species' leaves indicate the presence of dry structures. Using micromorphological studies, we analyzed the hairs of the studied species in terms of their shapes, sizes, and densities. We found some species have hairs with special appendages, which is due to the special conditions in which they have grown. Xeromorphic stomata were found on both leaf surfaces of all endemic gypsophyte plants of Semnan. Several studies have shown that gypsophytes have a specialized mechanism for regulating the absorption of sulfur and calcium from soils containing calcium sulfate or gypsum by their roots. The current study provides novel insights into the response of plant species to extreme conditions and potential adaptation strategies at micromorphological levels.

Flock House virus (FHV) is an animal virus and considered a model system for non-enveloped viruses. It has a small, icosahedral capsid (T=3) and a bipartite positive-sense RNA genome. We present an extensive study of the FHV capsid dynamics from all-atom molecular dynamics simulations of the complete capsid. The simulations explore different biologically relevant conditions (neutral/low pH, with/without RNA in the capsid) using the CHARMM force field. The results show that low pH destabilizes the capsid, causing radial expansion, and RNA stabilizes the capsid. The finding of low pH destabilization is biologically relevant because the capsid is exposed to low pH in the endosome, where conformational changes occur leading to genome release. We also observe structural changes at the fivefold and twofold symmetry axes that likely relate to the externalization of membrane active γ peptides through the fivefold vertex and extrusion of RNA at the twofold axis. Simulations using the Amber force field at neutral pH are also performed and display similar characteristics to the CHARMM simulations.

Force production in muscle is achieved through the interaction of myosin and actin. Strong binding states in active muscle are associated with Mg·ADP bound to the active site; release of Mg·ADP allows rebinding of ATP and dissociation from actin. Thus, Mg·ADP binding is positioned for adaptation as a force sensor. Mechanical loads on the lever arm can affect the ability of myosin to release Mg·ADP but exactly how this is done is poorly defined. Here we use F-actin decorated with double-headed smooth muscle myosin fragments in the presence of Mg·ADP to visualize the effect of internally supplied tension on the paired lever arms using cryoEM. The interaction of the paired heads with two adjacent actin subunits is predicted to place one lever arm under positive and the other under negative strain. The converter domain is believed to be the most flexible domain within myosin head. Our results, instead, point to the segment of heavy chain between the essential and regulatory light chains as the location of the largest structural change. Moreover, our results suggest no large changes in the myosin coiled coil tail as the locus of strain relief when both heads bind F-actin. The method would be adaptable to double-headed members of the myosin family. We anticipate that the study of actin-myosin interaction using double-headed fragments enables visualization of domains that are typically noisy in decoration with single-headed fragments.

Visualization of organelles and their interactions with other features in the native cell remains a challenge in modern biology. We have introduced cryo-scanning transmission electron tomography (CSTET), which can access 3D volumes on the scale of 1 micron with a resolution of nanometers, making it ideal for this task. Here we introduce two relevant advances: (a) we demonstrate the utility of multi-color super-resolution radial fluctuation light microscopy under cryogenic conditions (cryo-SRRF), and (b) we extend the use of deconvolution processing for dual-axis CSTET data. We show that cryo-SRRF nanoscopy is able to reach resolutions in the range of 100 nm, using commonly available fluorophores and a conventional widefield microscope for cryo-correlative light-electron microscopy. Such resolution aids in precisely identifying regions of interest before tomographic acquisition and enhances precision in localizing features of interest within the 3D reconstruction. Dual-axis CSTET tilt series data and application of entropy regularized deconvolution during post-processing results in close-to-isotropic resolution in the reconstruction without averaging. The integration of cryo-SRRF with deconvolved dual-axis CSTET provides a versatile workflow for studying unique objects in a cell.

AKT/PKB is a kinase involved in the regulation of a plethora of cell processes. Particularly, in embryonic stem cells (ESCs), AKT is crucial for the maintenance of pluripotency. Although the activation of this kinase relies on its recruitment to the cellular membrane and subsequent phosphorylation, multiple other post-translational modifications (PTMs), including SUMOylation, fine-tune its activity and target specificity. Since this PTM can also modify the localization and availability of different proteins, in this work we explored if SUMOylation impacts on the subcellular compartmentalization and distribution of AKT1 in ESCs. We found that this PTM does not affect AKT1 membrane recruitment, but it modifies the AKT1 nucleus/cytoplasm distribution, increasing its nuclear presence. Additionally, within this compartment, we found that AKT1 SUMOylation also impacts on the chromatin-binding dynamics of NANOG, a central pluripotency transcription factor. Remarkably, the oncogenic E17K AKT1 mutant produces major changes in all these parameters increasing the binding of NANOG to its targets, also in a SUMOylation dependent manner. These findings demonstrate that SUMOylation modulates AKT1 subcellular distribution, thus adding an extra layer of regulation of its function, possibly by affecting the specificity and interaction with its downstream targets.

Septins, often described as the fourth component of the cytoskeleton, are structural proteins found in a vast variety of living beings. They are related to small GTPases and thus, generally, present GTPase activity which may play an important (although incompletely understood) role in their organization and function. Septins polymerize into long non-polar filaments, in which each subunit interacts with two others by alternating interfaces, NC and G. In Saccharomyces cerevisiae four septins are organized in the following manner, [Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11]n in order to form filaments. Although septins were originally discovered in yeast and much is known regarding their biochemistry and function, only limited structural information about them is currently available. Here we present crystal structures of Cdc3/Cdc10 which provide the first view of the physiological interfaces formed by yeast septins. The G-interface has properties which place it in between that formed by SEPT2/SEPT6 and SEPT7/SEPT3 in human filaments. Switch I from Cdc10 contributes significantly to the interface, whereas in Cdc3 it is largely disorded. However, the significant negative charge density of the latter suggests it may have a unique role. At the NC-interface, we describe an elegant means by which the sidechain of a glutamine from helix α0 imitates a peptide group in order to retain hydrogen-bond continuity at the kink between helices α5 and α6 in the neighbouring subunit, thereby justifying the conservation of the helical distortion. Its absence from Cdc11, along with this structure's other unusual features are critically discussed by comparison with Cdc3 and Cdc10.

We report on the 3D ultrastructure of the mineralized petrous bone of mature pig using focused ion beam - scanning electron microscopy (FIB-SEM). We divide the petrous bone into two zones based on the degree of mineralization; one zone close to the otic chamber has higher mineral density than the second zone further away from the otic chamber. The hypermineralization of the petrous bone results in the collagen D-banding being poorly revealed in the lower mineral density zone (LMD), and absent in the high mineral density zone (HMD). We therefore could not use D-banding to decipher the 3D structure of the collagen assembly. Instead we exploited the anisotropy option in the Dragonfly image processing software to visualize the less mineralized collagen fibrils and/or nanopores that surround the more mineralized zones known as tesselles. This approach therefore indirectly tracks the orientations of the collagen fibrils in the matrix itself. We show that the HMD bone has a structure similar to that of woven bone, and the LMD is composed of lamellar bone with a plywood-like structural motif. This agrees with the fact that the bone close to the otic chamber is fetal bone and is not remodeled. The lamellar structure of the bone further away from the otic chamber is consistent with modeling/remodeling. The absence of the less mineralized collagen fibrils and nanopores resulting from the confluence of the mineral tesselles may contribute to shielding DNA during diagenesis. We show that anisotropy evaluation of the less mineralized collagen fibrils could be a useful tool to analyze bone ultrastructures and in particular the directionality of collagen fibril bundles that make up the bone matrix.

Titin is the largest protein found in nature and spans half a sarcomere in vertebrate striated muscle. The protein has multiple functions, including in the organisation of the thick filament and acting as a molecular spring during the muscle contraction cycle. Missense variants in titin have been linked to both cardiac and skeletal myopathies. Titin is primarily composed of tandem repeats of immunoglobulin and fibronectin type III (Fn3) domains in a variety of repeat patterns; however, the vast majority of these domains have not had their high-resolution structure determined experimentally. Here, we present the crystal structures of seven wild type titin Fn3 domains and two harbouring rare missense variants reported in hypertrophic cardiomyopathy (HCM) patients. All domains present the typical Fn3 fold, with the domains harbouring variants reported in HCM patients retaining the wild-type conformation. The effect on domain folding and stability were assessed for five rare missense variants found in HCM patients: four caused thermal destabilization of between 7 and 13 °C and one prevented the folding of its domain. The structures also allowed us to locate the positions of residues whose mutations have been linked to congenital myopathies and rationalise how they convey their deleterious effects. We find no evidence of physiological homodimer formation, excluding one hypothesised mechanism as to how titin variants could exert pathological effects.

Viral proteins interact with lipid membranes during various stages in the viral life cycle to propagate infection. p7 is an ion channel forming protein of Hepatitis C virus (HCV) that participates in viral assembly. Studies show that it has close ties to lipid metabolism in the cell and anionic phosphatidylserine (PS) lipids are suggested to be key for its permeabilizing function, but the mechanism of its interaction with the lipid environment is largely unknown. To begin unraveling the molecular processes of the protein, we evaluated the impact of lipid environment on the binding and insertion mechanism of p7 prior to channel formation and viral assembly using molecular dynamics simulations. It is seen that p7 is sensitive to its lipid environment and results in different remodeling patterns in membranes. Helix 1 (H1) is especially important for peptide insertion, with deeper entry taking place when the membrane contains phosphatidylserine (PS). Helix 2 (H2) and the adjacent loop connecting to Helix 3 (H3) prompts recruitment of phosphatidylethanolamine (PE) lipids to the protein binding site in membrane models with lower surface charge. This work provides perspectives on the interplay between protein-lipid dynamics and membrane composition, and insights on membrane reorganization in mechanisms of disease.