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Bacteriophage BPP-1 infects and kills Bordetella species that cause whooping cough. Its diversity-generating retroelement (DGR) provides a naturally occurring phage-display system, but engineering efforts are hampered without atomic structures. Here, we report a cryo electron microscopy structure of the BPP-1 head at 3.5 Å resolution. Our atomic model shows two of the three protein folds representing major viral lineages: jellyroll for its cement protein (CP) and HK97-like ('Johnson') for its major capsid protein (MCP). Strikingly, the fold topology of MCP is permuted non-circularly from the Johnson fold topology previously seen in viral and cellular proteins. We illustrate that the new topology is likely the only feasible alternative of the old topology. β-sheet augmentation and electrostatic interactions contribute to the formation of non-covalent chainmail in BPP-1, unlike covalent inter-protein linkages of the HK97 chainmail. Despite these complex interactions, the termini of both CP and MCP are ideally positioned for DGR-based phage-display engineering. DOI: http://dx.doi.org/10.7554/eLife.01299.001.

Mitochondrial complex III (CIII2) and complex IV (CIV), which can associate into a higher-order supercomplex (SC III2+IV), play key roles in respiration. However, structures of these plant complexes remain unknown. We present atomic models of CIII2, CIV, and SC III2+IV from Vigna radiata determined by single-particle cryoEM. The structures reveal plant-specific differences in the MPP domain of CIII2 and define the subunit composition of CIV. Conformational heterogeneity analysis of CIII2 revealed long-range, coordinated movements across the complex, as well as the motion of CIII2’s iron-sulfur head domain. The CIV structure suggests that, in plants, proton translocation does not occur via the H channel. The supercomplex interface differs significantly from that in yeast and bacteria in its interacting subunits, angle of approach and limited interactions in the mitochondrial matrix. These structures challenge long-standing assumptions about the plant complexes and generate new mechanistic hypotheses.

Atomic structures of mitochondrial enzyme complexes in plants are shedding light on their multiple functions.

"CryoEM" has come of age, enabling considerable structural insights into many facets of molecular biology. Here, we present a primer for microbiologists to understand the capabilities and limitations of two complementary cryoEM techniques for studying bacterial secretion systems. The first, single particle analysis, determines the structures of purified protein complexes to resolutions sufficient for molecular modeling, while the second, electron cryotomography and subtomogram averaging, tends to determine more modest resolution structures of protein complexes in intact cells. We illustrate these abilities with examples of insights provided into how secretion systems work by cryoEM, with a focus on type III secretion systems. [ABSTRACT FROM AUTHOR]

The RNA polymerase inhibitor favipiravir is currently in clinical trials as a treatment for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 A°. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, nonproductive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp, which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses. [ABSTRACT FROM AUTHOR]

Background: Identification and selection of protein particles in cryo-electron micrographs is an important step in single particle analysis. In this study, we developed a deep learning-based particle picking network to automatically detect particle centers from cryoEM micrographs. This is a challenging task due to the nature of cryoEM data, having low signal-to-noise ratios with variable particle sizes, shapes, distributions, grayscale variations as well as other undesirable artifacts. Results: We propose a double convolutional neural network (CNN) cascade for automated detection of particles in cryo-electron micrographs. This approach, entitled Deep Regression Picker Network or "DRPnet", is simple but very effective in recognizing different particle sizes, shapes, distributions and grayscale patterns corresponding to 2D views of 3D particles. Particles are detected by the first network, a fully convolutional regression network (FCRN), which maps the particle image to a continuous distance map that acts like a probability density function of particle centers. Particles identified by FCRN are further refined to reduce false particle detections by the second classification CNN. DRPnet's first CNN pretrained with only a single cryoEM dataset can be used to detect particles from different datasets without retraining. Compared to RELION template-based autopicking, DRPnet results in better particle picking performance with drastically reduced user interactions and processing time. DRPnet also outperforms the state-of-the-art particle picking networks in terms of the supervised detection evaluation metrics recall, precision, and F-measure. To further highlight quality of the picked particle sets, we compute and present additional performance metrics assessing the resulting 3D reconstructions such as number of 2D class averages, efficiency/angular coverage, Rosenthal-Henderson plots and local/global 3D reconstruction resolution. Conclusion: DRPnet shows greatly improved time-savings to generate an initial particle dataset compared to manual picking, followed by template-based autopicking. Compared to other networks, DRPnet has equivalent or better performance. DRPnet excels on cryoEM datasets that have low contrast or clumped particles. Evaluating other performance metrics, DRPnet is useful for higher resolution 3D reconstructions with decreased particle numbers or unknown symmetry, detecting particles with better angular orientation coverage. [ABSTRACT FROM AUTHOR]

We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications. [ABSTRACT FROM AUTHOR]

CryoEM has become the method of choice for determining the structure of large macromolecular complexes in multiple conformations, at resolutions where unambiguous atomic models can be built. Two effects that have limited progress in single-particle cryoEM are (i) beam-induced movement during image acquisition and (ii) protein adsorption and denaturation at the air-water interface during specimen preparation. While beam-induced movement now appears to have been resolved by all-gold specimen support grids with very small holes, surface effects at the air-water interface are a persistent problem. Strategies to overcome these effects include the use of alternative support films and new techniques for specimen deposition. We examine the future potential of recording perfect images of biological samples for routine structure determination at atomic resolution. [ABSTRACT FROM AUTHOR]

Leginon is a system for automated data acquisition from a transmission electron microscope. Here we provide an updated summary of the overall Leginon architecture and an update of the current state of the package. We also highlight a few recent developments to provide some concrete examples and use cases. [ABSTRACT FROM AUTHOR]

The P4 ATPases use ATP hydrolysis to transport large lipid substrates across lipid bilayers. The structures of the endosome- and Golgi-localized phosphatidylserine flippases—such as the yeast Drs2 and human ATP8A1—have recently been reported. However, a substrate-binding site on the cytosolic side has not been found, and the transport mechanisms of P4 ATPases with other substrates are unknown. Here, we report structures of the S. cerevisiae Dnf1–Lem3 and Dnf2–Lem3 complexes. We captured substrate phosphatidylcholine molecules on both the exoplasmic and cytosolic sides and found that they have similar structures. Unexpectedly, Lem3 contributes to substrate binding. The conformational transitions of these phosphatidylcholine transporters match those of the phosphatidylserine transporters, suggesting a conserved mechanism among P4 ATPases. Dnf1/Dnf2 have a unique P domain helix-turn-helix insertion that is important for function. Therefore, P4 ATPases may have retained an overall transport mechanism while evolving distinct features for different lipid substrates.

The yeast THO complex is recruited to active genes and interacts with the RNA-dependent ATPase Sub2 to facilitate the formation of mature export-competent messenger ribonucleoprotein particles and to prevent the co-transcriptional formation of RNA:DNA-hybrid-containing structures. How THO-containing complexes function at the mechanistic level is unclear. Here, we elucidated a 3.4 Å resolution structure of Saccharomyces cerevisiae THO-Sub2 by cryo-electron microscopy. THO subunits Tho2 and Hpr1 intertwine to form a platform that is bound by Mft1, Thp2, and Tex1. The resulting complex homodimerizes in an asymmetric fashion, with a Sub2 molecule attached to each protomer. The homodimerization interfaces serve as a fulcrum for a seesaw-like movement concomitant with conformational changes of the Sub2 ATPase. The overall structural architecture and topology suggest the molecular mechanisms of nucleic acid remodeling during mRNA biogenesis.

The discovery of Mas-related G protein-coupled receptors (MRGPRs) in itch sensation promised a search for novel therapeutics of itch that ultimately met with little success. Recent structural determination of these receptors by Roth and Sun marks a big step forward in the search for therapeutics of debilitating itch. [ABSTRACT FROM AUTHOR]

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of major facilitator superfamily transporters. With a conformation-selective nanobody, we determined a low-sugar affinity inward-facing Na + -bound cryoEM structure. The available outward-facing sugar-bound structures showed that the N- and C-terminal residues of the inner barrier contribute to the sugar selectivity. The inward-open conformation shows that the sugar selectivity pocket is also broken when the inner barrier is broken. Isothermal titration calorimetry measurements revealed that this inward-facing conformation trapped by this nanobody exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is supported by molecular dynamics simulations. Furthermore, the hydron/deuterium exchange mass spectrometry analyses allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport., Competing Interests: PH, SK, KW, NB, PM, AB, SS, EP, HK, RL, JS, LG No competing interests declared, YS, RV employee of Thermo Fisher Scientific, (© 2023, Hariharan et al.)

Heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) is a human ribonucleoprotein that transports RNA to designated locations for translation via its ability to phase separate. Its mutated form, D290V, is implicated in multisystem proteinopathy known to afflict two families, mainly with myopathy and Paget's disease of bone. Here, we investigate this mutant form of hnRNPA2 by determining cryo-EM structures of the recombinant D290V low complexity domain. We find that the mutant form of hnRNPA2 differs from the WT fibrils in four ways. In contrast to the WT fibrils, the PY-nuclear localization signals in the fibril cores of all three mutant polymorphs are less accessible to chaperones. Also, the mutant fibrils are more stable than WT fibrils as judged by phase separation, thermal stability, and energetic calculations. Similar to other pathogenic amyloids, the mutant fibrils are polymorphic. Thus, these structures offer evidence to explain how a D-to-V missense mutation diverts the assembly of reversible, functional amyloid-like fibrils into the assembly of pathogenic amyloid, and may shed light on analogous conversions occurring in other ribonucleoproteins that lead to neurological diseases such as amyotrophic lateral sclerosis and frontotemporal dementia., Competing Interests: Conflict of interest D. S. E. is an advisor and equity shareholder in ADRx, Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Exonuclease VII (ExoVII) is a ubiquitous bacterial nuclease. Encoded by the xseA and xseB genes, ExoVII participates in multiple nucleic acid-dependent pathways including the processing of multicopy single-stranded DNA and the repair of covalent DNA-protein crosslinks (DPCs). Although many biochemical properties of ExoVII have been defined, little is known about its structure/function relationships. Here, we use cryoelectron microscopy (cryoEM) to determine that Escherichia coli ExoVII comprises a highly elongated XseA 4 ·XseB 24 holo-complex. Each XseA subunit dimerizes through a central extended α-helical segment decorated by six XseB subunits and a C-terminal, domain-swapped β-barrel element; two XseA 2 ·XseB 12 subcomplexes further associate using N-terminal OB (oligonucleotide/oligosaccharide-binding) folds and catalytic domains to form a spindle-shaped, catenated octaicosamer. The catalytic domains of XseA, which adopt a nuclease fold related to 3-dehydroquinate dehydratases, are sequestered in the center of the complex and accessible only through large pores formed between XseA tetramers. The architectural organization of ExoVII, combined with biochemical studies, indicate that substrate selectivity is controlled by steric access to its nuclease elements and that tetramer dissociation results from substrate DNA binding. Despite a lack of sequence and fold homology, the physical organization of ExoVII is reminiscent of Mre11·Rad50/SbcCD ATP (adenosine triphosphate)-dependent nucleases used in the repair of double-stranded DNA breaks, including those formed by DPCs through aberrant topoisomerase activity, suggesting that there may have been convergent evolutionary pressure to contend with such damage events., Competing Interests: Competing interests statement:The authors declare no competing interest.

Surface layers (S-layers) are resilient two-dimensional protein lattices that encapsulate many bacteria and most archaea. In archaea, S-layers usually form the only structural component of the cell wall and thus act as the final frontier between the cell and its environment. Therefore, S-layers are crucial for supporting microbial life. Notwithstanding their importance, little is known about archaeal S-layers at the atomic level. Here, we combined single-particle cryo electron microscopy, cryo electron tomography, and Alphafold2 predictions to generate an atomic model of the two-component S-layer of Sulfolobus acidocaldarius . The outer component of this S-layer (SlaA) is a flexible, highly glycosylated, and stable protein. Together with the inner and membrane-bound component (SlaB), they assemble into a porous and interwoven lattice. We hypothesise that jackknife-like conformational changes in SlaA play important roles in S-layer assembly., Competing Interests: LG, MM, RC, KS, MG, LC, VG, DK, MS, CH, MI, BD No competing interests declared, (© 2024, Gambelli et al.)

Super-resolution cryo-correlative light and electron microscopy (SRcryoCLEM) is emerging as a powerful method to enable targeted in situ structural studies of biological samples. By combining the high specificity and localization accuracy of single-molecule localization microscopy (cryoSMLM) with the high resolution of cryo-electron tomography (cryoET), this method enables accurately targeted data acquisition and the observation and identification of biomolecules within their natural cellular context. Despite its potential, the adaptation of SRcryoCLEM has been hindered by the need for specialized equipment and expertise. In this chapter, we outline a workflow for cryoSMLM and cryoET-based SRcryoCLEM, and we demonstrate that, given the right tools, it is possible to incorporate cryoSMLM into an established cryoET workflow. Using Vimentin as an exemplary target of interest, we demonstrate all stages of an SRcryoCLEM experiment: performing cryoSMLM, targeting cryoET acquisition based on single-molecule localization maps, and correlation of cryoSMLM and cryoET datasets using scNodes, a software package dedicated to SRcryoCLEM. By showing how SRcryoCLEM enables the imaging of specific intracellular components in situ, we hope to facilitate adoption of the technique within the field of cryoEM., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

The packaging of DNA into preformed capsids is a critical step during herpesvirus infection. For herpes simplex virus, this process requires the products of seven viral genes: the terminase proteins pUL15, pUL28, and pUL33; the capsid vertex-specific component (CVSC) proteins pUL17 and pUL25; and the portal proteins pUL6 and pUL32. The pUL6 portal dodecamer is anchored at one vertex of the capsid by interactions with the adjacent triplexes as well as helical density attributed to the pUL17 and pUL25 subunits of the CVSC. To define the roles and structures of the CVSC proteins in virus assembly and DNA packaging, we isolated a number of recombinant viruses expressing pUL25, pUL17, and pUL36 fused with green or red fluorescent proteins as well as viruses with specific deletions in the CVSC genes. Biochemical and structural studies of these mutants demonstrated that (i) four of the helices in the CVSC helix bundle can be attributed to two copies each of pUL36 and pUL25, (ii) pUL17 and pUL6 are required for capsid binding of the terminase complex in the nucleus, (iii) pUL17 is important for determining the site of the first cleavage reaction generating replicated genomes with termini derived from the long-arm component of the herpes simplex virus 1 (HSV-1) genome, (iv) pUL36 serves no direct role in cleavage/packaging, (v) cleavage and stable packaging of the viral genome involve an ordered interaction of the terminase complex and pUL25 with pUL17 at the portal vertex, and (vi) packaging of the viral genome results in a dramatic displacement of the portal. [ABSTRACT FROM AUTHOR]

Blotting times for conventional cryoEM specimen preparation complicate time‐resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air–water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass‐fiber filter held against the opposite side, often called the 'back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through‐grid wicking were combined in a high‐speed specimen‐preparation device that was named 'Back‐it‐up' or BIU. The high liquid‐absorption capacity of the glass fiber compared with self‐wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through‐grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent‐solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations. [ABSTRACT FROM AUTHOR]

Background: The insulin receptor-related receptor (IRR) was originally discovered due to its high homology to the other family members (insulin receptor and insulin-like growth factor 1 receptor). We determined that IRR can be activated by mildly alkaline extracellular media and has typical features of the ligand-receptor interaction, including its specificity and dose-dependence. Since pH-sensitive properties of IRR are determined by its ectodomain; therefore, we chose as an option to study the soluble extracellular domain IRR. Methods: The investigation carried out in Titan Krios 60-300 TEM/STEM (FEI, USA) CryoEM, equipped with direct electron detector Falcon II (FEI, USA) and Cs image corrector (CEOS, Germany), at an accelerating voltage of 300 kV. Data processing and 3D reconstruction were carried out using computing resources of the Federal Collective Usage Center Complex for Simulation and Data Processing for Mega-Science Facilities at NRC “Kurchatov Institute.” Results: The obtained 2D classifications of particles of the ectodomain IRR at a neutral pH form several 3D models. This indicates that the ectodomain has several possible conformations, which is consistent with our previously obtained data using SEC-SAXS and AFM. In the future, additional careful data processing is required, as well as studies of the IRR ectodomain in mildly alkaline pH. Conclusion: In this study, we presented the structural characteristics of the IRR ectodomain obtained by CryoEM. These results are an important step towards understanding the mechanism of functioning of the IRR.

Coat proteins of tailed, dsDNA phages and in herpesviruses include a conserved core similar to the bacteriophage HK97 subunit. This core is often embellished with other domains such as the telokin Ig-like domain of phage P22. Eighty-six P22-like phages and prophages with sequenced genomes share a similar set of virion assembly genes and, based on comparisons of twelve viral assembly proteins (structural and assembly/packaging chaperones), these phages are classified into three groups (P22-like, Sf6-like, and CUS-3-like). We used cryo-electron microscopy and 3D image reconstruction to determine the structures of Sf6 procapsids and virions (~7Å resolution), and the structure of the entire, asymmetric Sf6 virion (16-Å resolution). The Sf6 coat protein is similar to that of P22 yet it has differences in the telokin domain and in its overall quaternary organization. Thermal stability and agarose gel experiments show that Sf6 virions are slightly less stable than those of P22. Finally, bacterial host outer membrane proteins A and C were identified in lipid vesicles that co-purify with Sf6 particles, but are not components of the capsid.

MicroED has recently emerged as a powerful method for the analysis of biological structures at atomic resolution. This technique has been largely limited to protein nanocrystals which grow either as needles or plates measuring only a few hundred nanometers in thickness. Furthermore, traditional microED data processing uses established X-ray crystallography software that is not optimized for handling compound effects that are unique to electron diffraction data. Here, we present an integrated workflow for microED, from sample preparation by cryo-focused ion beam milling, through data collection with a standard Ceta-D detector, to data processing using the DIALS software suite, thus enabling routine atomic structure determination of protein crystals of any size and shape using microED. We demonstrate the effectiveness of the workflow by determining the structure of proteinase K to 2.0 Å resolution and show the advantage of using protein crystal lamellae over nanocrystals.

Genome replication is initiated from specific origin sites established by dynamic events. The Origin Recognition Complex (ORC) is necessary for orchestrating the initiation process by binding to origin DNA, recruiting CDC6, and assembling the MCM replicative helicase on DNA. Here we report five cryoEM structures of the human ORC (HsORC) that illustrate the native flexibility of the complex. The absence of ORC1 revealed a compact, stable complex of ORC2-5. Introduction of ORC1 opens the complex into several dynamic conformations. Two structures revealed dynamic movements of the ORC1 AAA+ and ORC2 winged-helix domains that likely impact DNA incorporation into the ORC core. Additional twist and pinch motions were observed in an open ORC conformation revealing a hinge at the ORC5·ORC3 interface that may facilitate ORC binding to DNA. Finally, a structure of ORC was determined with endogenous DNA bound in the core revealing important differences between human and yeast origin recognition.

G-protein-gated inward rectifier potassium (GIRK) channels are regulated by G proteins and PIP2. Here, using cryo-EM single particle analysis we describe the equilibrium ensemble of structures of neuronal GIRK2 as a function of the C8-PIP2 concentration. We find that PIP2 shifts the equilibrium between two distinguishable structures of neuronal GIRK (GIRK2), extended and docked, towards the docked form. In the docked form the cytoplasmic domain, to which Gβγ binds, becomes accessible to the cytoplasmic membrane surface where Gβγ resides. Furthermore, PIP2 binding reshapes the Gβγ binding surface on the cytoplasmic domain, preparing it to receive Gβγ. We find that cardiac GIRK (GIRK1/4) can also exist in both extended and docked conformations. These findings lead us to conclude that PIP2 influences GIRK channels in a structurally similar manner to Kir2.2 channels. In Kir2.2 channels, the PIP2-induced conformational changes open the pore. In GIRK channels, they prepare the channel for activation by Gβγ.

Resolution (global and local) is one of the most reported metrics of quality measurement in Single Particle Analysis (SPA). However, in electron tomography, the situation is different and its computation is not straightforward. Typically, resolution estimation is global and, therefore, reduces the assessment of a whole tomogram to a single number. However, it is known that tomogram quality is spatially variant. Still, up to our knowledge, a method to estimate local quality metrics in tomography is lacking. This work introduces MonoTomo, a method developed to estimate locally in a tomogram the highest reliable frequency component, expressed as a form of local resolution. The fundamentals lie in a local analysis of the density map via monogenic signals, which, in analogy to MonoRes, allows for local estimations. Results with experimental data show that the local resolution range that MonoTomo casts agrees with reported resolution values for experimental data sets, with the advantage of providing a local estimation. A range of applications of MonoTomo are suggested for further exploration.

The ClpXP degradation machine consists of a hexameric AAA+ unfoldase (ClpX) and a pair of heptameric serine protease rings (ClpP) that unfold, translocate, and subsequently degrade client proteins. ClpXP is an important target for drug development against infectious diseases. Although structures are available for isolated ClpX and ClpP rings, it remains unknown how symmetry mismatched ClpX and ClpP work in tandem for processive substrate translocation into the ClpP proteolytic chamber. Here, we present cryo-EM structures of the substrate-bound ClpXP complex from Neisseria meningitidis at 2.3 to 3.3 Å resolution. The structures allow development of a model in which the sequential hydrolysis of ATP is coupled to motions of ClpX loops that lead to directional substrate translocation and ClpX rotation relative to ClpP. Our data add to the growing body of evidence that AAA+ molecular machines generate translocating forces by a common mechanism.

The peptidase-containing ATP-binding cassette transporters (PCATs) are unique members of the ABC transporter family that proteolytically process and export peptides and proteins. Each PCAT contains two peptidase domains that cleave off the secretion signal, two transmembrane domains forming a translocation pathway, and two nucleotide-binding domains that hydrolyze ATP. Previously the crystal structures of a PCAT from Clostridium thermocellum (PCAT1) were determined in the absence and presence of ATP, revealing how ATP binding regulates the protease activity and access to the translocation pathway. However, how the substrate CtA, a 90-residue polypeptide, is recognized by PCAT1 remained elusive. To address this question, we determined the structure of the PCAT1-CtA complex by electron cryo-microscopy (cryo-EM) to 3.4 Å resolution. The structure shows that two CtAs are bound via their N-terminal leader peptides, but only one is positioned for cleavage and translocation. Based on these results, we propose a model of how substrate cleavage, ATP hydrolysis, and substrate translocation are coordinated in a transport cycle.

The activation mechanism of the nitric oxide receptor has been revealed by cryo-electron microscopy.

The synaptonemal complex (SC) keeps homologous chromosomes in close alignment during meiotic recombination. A hallmark of the SC is the presence of its constituent protein SYCP3 on the chromosome axis. During SC assembly, SYCP3 is deposited on both axes of the homologue pair, forming axial elements that fuse into the lateral element (LE) in the tripartite structure of the mature SC. We have used cryo-electron tomography and atomic force microscopy to study the mechanism of assembly and DNA binding of the SYCP3 fibre. We find that the three-dimensional architecture of the fibre is built on a highly irregular arrangement of SYCP3 molecules displaying very limited local geometry. Interaction between SYCP3 molecules is driven by the intrinsically disordered tails of the protein, with no contact between the helical cores, resulting in a flexible fibre assembly. We demonstrate that the SYCP3 fibre can engage in extensive interactions with DNA, indicative of an efficient mechanism for incorporation of DNA within the fibre. Our findings suggest that SYCP3 deposition on the chromosome axis might take place by polymerization into a fibre that is fastened to the chromosome surface via DNA binding.

Retroviruses have a very complex and tightly controlled life cycle which has been studied intensely for decades. After a virus enters the cell, it reverse-transcribes its genome, which is then integrated into the host genome, and subsequently all structural and regulatory proteins are transcribed and translated. The proteins, along with the viral genome, assemble into a new virion, which buds off the host cell and matures into a newly infectious virion. If any one of these steps are faulty, the virus cannot produce infectious viral progeny. Recent advances in structural and molecular techniques have made it possible to better understand this class of viruses, including details about how they regulate and coordinate the different steps of the virus life cycle. In this review we summarize the molecular analysis of the assembly and maturation steps of the life cycle by providing an overview on structural and biochemical studies to understand these processes. We also outline the differences between various retrovirus families with regards to these processes.

During the crystal structure analysis of an ATP‐binding cassette (ABC) transporter overexpressed in Escherichia coli, a contaminant protein was crystallized. The identity of the contaminant was revealed by mass spectrometry to be the Escherichia coli transcription terminator factor Rho, structures of which had been previously determined in different conformational states. Although Rho was present at only ∼1% of the target protein (a bacterial homolog of the eukaryotic ABC transporter of mitochondria from Novosphingobium aromaticivorans; NaAtm1), it preferentially crystallized in space group C2 as thin plates that diffracted to 3.30 Å resolution. The structure of Rho in this crystal form exhibits a hexameric open‐ring staircase conformation with bound ATP; this characteristic structure was also observed on electron‐microscopy grids of the NaAtm1 preparation. [ABSTRACT FROM AUTHOR]

Structure determination of novel biological macromolecules by X‐ray crystallography can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective search models for molecular replacement to overcome the phase problem. Independence from the need for a complete pre‐existing model with sequence similarity to the crystallized molecule is the primary appeal of ARCIMBOLDO, a suite of programs which employs this ab initio algorithm for phase determination. Here, the use of ARCIMBOLDO is investigated to overcome the phase problem with the electron cryomicroscopy (cryoEM) method known as microcrystal electron diffraction (MicroED). The results support the use of the ARCIMBOLDO_SHREDDER pipeline to provide phasing solutions for a structure of proteinase K from 1.6 Å resolution data using model fragments derived from the structures of proteins sharing a sequence identity of as low as 20%. ARCIMBOLDO_SHREDDER identified the most accurate polyalanine fragments from a set of distantly related sequence homologues. Alternatively, such templates were extracted in spherical volumes and given internal degrees of freedom to refine towards the target structure. Both modes relied on the rotation function in Phaser to identify or refine fragment models and its translation function to place them. Model completion from the placed fragments proceeded through phase combination of partial solutions and/or density modification and main‐chain autotracing using SHELXE. The combined set of fragments was sufficient to arrive at a solution that resembled that determined by conventional molecular replacement using the known target structure as a search model. This approach obviates the need for a single, complete and highly accurate search model when phasing MicroED data, and permits the evaluation of large fragment libraries for this purpose. [ABSTRACT FROM AUTHOR]

Cellular studies of filamentous actin (F‐actin) processes commonly utilize fluorescent versions of toxins, peptides, and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F‐actin markers for a particular requirement. Here, we describe the electron cryomicroscopy structures of phalloidin, lifeAct, and utrophin bound to F‐actin, providing a comprehensive high‐resolution structural comparison of widely used actin markers and their influence towards F‐actin. Our results show that phalloidin binding does not induce specific conformational change and lifeAct specifically recognizes closed D‐loop conformation, i.e., ADP‐Pi or ADP states of F‐actin. The structural models aided designing of minimal utrophin and a shorter lifeAct, which can be utilized as F‐actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin‐binding proteins and thus offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants. Synopsis: Study of the cellular actin cytoskeleton relies on fluorescent probes based on toxins (phalloidin), peptides (LifeAct), or actin‐binding proteins (e.g. utrophin). Cryo‐EM structures and comparative analyses of these three probes bound to actin filaments offer guidance for their respective suitabilities. Phalloidin binding to actin filament does not induce actin conformational changes.LifeAct preferentially binds to the closed D‐loop conformation of actin filaments.The utrophin CH1 domain is sufficient for interaction with F‐actin. [ABSTRACT FROM AUTHOR]

The "omics revolution: beyond genomics" satellite meeting, run under the auspices of the Australian Peptide Association, The Human Proteome Organisation (HUPO) and the HUPO Australia/New Zealand Chromosome 7 initiative, was held at the Oaks Resort, Port Douglas, Queensland, Australia, on 8th September 2019, immediately prior to the 13th Australian Peptide Conference. The meeting, which had over 100 participants representing Australasia, Europe and America, focused on recent advances in omics-related technologies, including mass spectrometry, biosensors and CryoEM, which will assist in the clinical translation of proteomics towards precision/personalized medicine. An overview of the conference and a summary of the oral presentations are presented. [ABSTRACT FROM AUTHOR]

The last 20 years have seen incredible advances in electron microscopy (EM). Cryoelectron microscopy can now resolve protein structures to a previously unimaginable resolution but the advances in cellular EM are just as significant. I will take this opportunity to briefly summarize some of the new developments in cellular EM. [ABSTRACT FROM AUTHOR]

Cryoelectron microscopy (cryo-EM) has provided unprecedented insights into amyloid fibril structures, including those associated with disease. However, these structures represent the endpoints of long assembly processes, and their relationship to fibrils formed early in assembly is unknown. Consequently, whether different fibril architectures, with potentially different pathological properties, form during assembly remains unknown. Here, we used cryo-EM to determine structures of amyloid fibrils at different times during in vitro fibrillation of a disease-related variant of human islet amyloid polypeptide (IAPP-S20G). Strikingly, the fibrils formed in the lag, growth, and plateau phases have different structures, with new forms appearing and others disappearing as fibrillation proceeds. A time course with wild-type hIAPP also shows fibrils changing with time, suggesting that this is a general property of IAPP amyloid assembly. The observation of transiently populated fibril structures has implications for understanding amyloid assembly mechanisms with potential new insights into amyloid progression in disease. [Display omitted] • IAPP-S20G amyloid fibril polymorphism drastically changes as assembly progresses • Different fibril structures populate the lag, growth, and stationary phases of assembly • Early populations contain transient species that disappear by the end of the reaction • Structural lineages are proposed from common fibril cores in increasing assembly sizes Cryo-EM sampling of a diabetes-associated variant of islet amyloid polypeptide during the lag, growth, and stationary phases of fibril assembly reveals the dynamic nature of amyloid polymorphism over time. [ABSTRACT FROM AUTHOR]

Lymphocyte activation gene 3 protein (LAG3) is an inhibitory receptor that is upregulated on exhausted T cells in tumors. LAG3 is a major target for cancer immunotherapy with many anti-LAG3 antibodies in clinical trials. However, there is no structural information on the epitopes recognized by these antibodies. We determined the single-particle cryoEM structure of a therapeutic antibody (favezelimab) bound to LAG3 to 3.5 Å resolution, revealing that favezelimab targets the LAG3-binding site for MHC class II, its canonical ligand. The small size of the complex between the conventional (monovalent) Fab of favezelimab and LAG3 (∼100 kDa) presented a challenge for cryoEM. Accordingly, we engineered a bivalent version of Fab favezelimab that doubled the size of the Fab–LAG3 complex and conferred a highly identifiable shape to the complex that facilitated particle selection and orientation for image processing. This study establishes bivalent Fabs as new fiducial markers for cryoEM analysis of small proteins. [Display omitted] • LAG3 is a major target for cancer immunotherapy using anti-LAG3 antibodies • We determined the cryoEM structure of LAG3 bound to a therapeutic antibody • Structure determination required engineering a bivalent Fab to increase complex size • Bivalent Fabs are new fiducial markers for cryoEM analysis of small proteins LAG3 is receptor on T cells that is a major target for cancer immunotherapy using monoclonal antibodies against LAG3. However, it is unknown how these antibodies bind LAG3. Mishra et al. used cryoEM to identify the site on LAG3 targeted by a therapeutic antibody, which explains how the antibody works. [ABSTRACT FROM AUTHOR]

Background: Yellow fever virus (YFV) is the prototype virus of the genus Flavivirus. It is endemic to sub-Saharan Africa and tropical South America. YF disease ranges from asymptomatic to severe jaundice and hemorrhagic fever. The flavivirus virion core is enveloped by a lipid membrane with integrated membrane (M) proteins and envelope (E) proteins that form the outer surface of the virion. The Е protein provides stability to the viral particle and is responsible for early infection stages. Flaviviruses are heterogeneous in nature, which is related to their maturation process. Samples always contain mature, immature, half-mature, and damaged particles. Thus, cryo-EM is a method of choice for their structure determination. During the early stages of cryo-EM development, structures of flaviviruses were studied at 10–20 Å resolution. However, due to the progress of recent years, it became possible to determine flavivirus structures at a resolution of 5.6-2.6 Å. The cryo-EM method was used to obtain structural data of virions of dengue fever virus, Zika virus, TBE virus, etc. For YFV, only the cryo-EM structure of the immature virions at a low resolution of 25 Å was determined (Y. Zhang et al. 2003 doi:10.1093/emboj/cdg270). However, the structure of mature (most infectious) YFV particles is still unknown. Methods: Virus sample was produced in Vero cell culture. YFV-17D was inactivated and purified using ultracentrifugation. The concentration of viral particles in the target inactivated YFV-17D (iYFV-17D) was evaluated by spectrophotometry and by estimating the concentration of E protein determined by PAGE electrophoresis. Preliminary quality control of iYFV-17D sample was performed using negative staining TEM. Cryo-EM data were collected using cryo-TEM Krios (Thermo-Fisher, USA) at 300kV using DED Falcon II. Dataset was preprocessed using Warp. Further processing was performed in Relion 3.1 and CisTEM. Model building was carried out using Isolde, Phenix and Coot software. Results: A protocol of production and purification of highly concentrated (~2x1012) monodisperse inactivated iYFV-17D sample was developed. Cryo-EM structure of mature iYFV was solved at 4.1 Å resolution. The structure of YFV is similar to other known flavivirus structures, with 180 copies of protein E arranged in a herringbone pattern that makes up the icosahedral shell. Conclusion: The high-resolution structure of mature iYFV-17D allowed to elucidate special features of this flavivirus and may be useful for vaccine improvement and drug development.

Soluble guanylate cyclase (sGC) is the primary receptor for nitric oxide (NO) in mammalian nitric oxide signaling. We determined structures of full-length Manduca sexta sGC in both inactive and active states using cryo-electron microscopy. NO and the sGC-specific stimulator YC-1 induce a 71° rotation of the heme-binding β H-NOX and PAS domains. Repositioning of the β H-NOX domain leads to a straightening of the coiled-coil domains, which, in turn, use the motion to move the catalytic domains into an active conformation. YC-1 binds directly between the β H-NOX domain and the two CC domains. The structural elongation of the particle observed in cryo-EM was corroborated in solution using small angle X-ray scattering (SAXS). These structures delineate the endpoints of the allosteric transition responsible for the major cyclic GMP-dependent physiological effects of NO.