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Dyneins are motor proteins responsible for transport in the cytoplasm and the beating of axonemes in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a coiled-coil stalk. We address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using helical Relion. The majority of the MTBD is rigid upon binding, with the transition to the high-affinity state controlled by the movement of a single helix at the MTBD interface. DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that contacts the adjacent protofilament. Unexpectedly, we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.

Background: Membrane complexes are of great importance for cell functioning. Among the best studied complexes are rotor ATPases which create ATP from ADP, or vice versa (J.E. Walker, Biochem. Soc. Trans. 41, pp. 1-16, 2013). Generally, rotor ATPases consist of a soluble part and a membrane-embedded part. The membrane part includes a c-ring – a symmetric oligomer of subunits c, which is rotating in the membrane during the protein operation. The inside pore of the c-rings in some cases is plugged by phospholipids. While the protein components are well ordered in the structures, the surrounding lipid molecules, including those trapped inside the c-ring, usually are not resolved. Most probably this is due to the great flexibility of lipids (P. Buslaev et al., J. Chem. Theory Compyt. 12, pp. 1019-1028, 2017) and lack of specific lipid-protein interactions, or due to techniques used for sample preparation. Recently, a Cryo-EM structure of spinach chloroplast ATPase with recognizable densities in the membrane region has become available, providing an opportunity to compare the modeled lipid positions with the experimental data (A. Hahn et al., Science eaat4318, 2018). Methods: We introduce nature-inspired approach to model the lipids inside the c-ring. It uses a biasing force to assemble the whole ring, essentially by incorporating experimental restraints into the coarse grained (CG) molecular dynamics (MD) simulation. The numerical comparison of modeled lipid densities with the EM maps was performed using the real space correlation coefficient (RSCC). Results: The structures converged into an assembled ring. The numbers of lipids trapped at the loop side and the NC-side were consistent in different runs: 9 to 11 and 13 to 15, respectively. However, RSCC of modeled lipid densities with the EM map was poor. Thus, we conducted short CG simulations where lipids were removed one-by-one to maximize RSCC (Fig. 1). The best fit (RSCC > 0.8) was observed for a system with 6 lipids at the loop side and 9 lipids at the NC side. The obtained systems were converted to atomistic models and were stable for several hundreds of nanoseconds. The atomistic models also correlated with experimental data well (RSCC > 0.8) and showed the same trend as densities for coarse grained simulations. Conclusion: We expect that the approach will be helpful for modelling of the lipids encapsulated within membrane protein and for studies of assembly of membrane complexes.

The SNF2h remodeler slides nucleosomes most efficiently as a dimer, yet how the two protomers avoid a tug-of-war is unclear. Furthermore, SNF2h couples histone octamer deformation to nucleosome sliding, but the underlying structural basis remains unknown. Here we present cryo-EM structures of SNF2h-nucleosome complexes with ADP-BeFx that capture two potential reaction intermediates. In one structure, histone residues near the dyad and in the H2A-H2B acidic patch, distal to the active SNF2h protomer, appear disordered. The disordered acidic patch is expected to inhibit the second SNF2h protomer, while disorder near the dyad is expected to promote DNA translocation. The other structure doesn’t show octamer deformation, but surprisingly shows a 2 bp translocation. FRET studies indicate that ADP-BeFx predisposes SNF2h-nucleosome complexes for an elemental translocation step. We propose a model for allosteric control through the nucleosome, where one SNF2h protomer promotes asymmetric octamer deformation to inhibit the second protomer, while stimulating directional DNA translocation.

Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions face the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.

Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems., Competing Interests: Competing interests statement:T.W. and M.M.E.-G. are employees of YPS, Ltd. J.D. was an employee of JEOL, UK during this work. M.M. and C.S.-B. are employees of DECTRIS, Ltd. G.M., R.H., and C.J.R. are inventors on a patent application EP No. 20789961.8 filed by UKRI related to this work.

Importance: A new class of bacterial protein sensors monitors intracellular levels of S -adenosylmethionine to modulate cell morphology, chemotaxis, and biofilm formation. Simultaneous regulation of these behaviors enables bacterial pathogens to survive within their niche. This sensor, exemplified by Treponema denticola CheWS, is anchored to the chemotaxis array and its sensor domain is located below the chemotaxis rings. This position may allow the sensor to directly interact with the chemotaxis histidine kinase CheA. Collectively, these data establish a critical role of CheWS in pathogenesis and further illustrate the impact of studying non-canonical chemotaxis proteins., Competing Interests: The authors declare no conflict of interest.

Importance: Bacterial chemotaxis is a ubiquitous behavior that enables cell movement toward or away from specific chemicals. It serves as an important model for understanding cell sensory signal transduction and motility. Characterization of the molecular mechanisms underlying chemotaxis is of fundamental interest and requires a high-resolution structural picture of the sensing machinery, the chemosensory array. In this study, we combine cryo-electron tomography and molecular simulation to present the complete structure of the core signaling unit, the basic building block of chemosensory arrays, from Escherichia coli . Our results provide new insight into previously poorly-resolved regions of the complex and offer a structural basis for designing new experiments to test mechanistic hypotheses., Competing Interests: The authors declare no conflict of interest.

Energy conversion by electron transport chains occurs through the sequential transfer of electrons between protein complexes and intermediate electron carriers, creating the proton motive force that enables ATP synthesis and membrane transport. These protein complexes can also form higher order assemblies known as respiratory supercomplexes (SCs). The electron transport chain of the opportunistic pathogen Pseudomonas aeruginosa is closely linked with its ability to invade host tissue, tolerate harsh conditions, and resist antibiotics but is poorly characterized. Here, we determine the structure of a P. aeruginosa SC that forms between the quinol:cytochrome c oxidoreductase (cytochrome bc 1 ) and one of the organism's terminal oxidases, cytochrome cbb 3 , which is found only in some bacteria. Remarkably, the SC structure also includes two intermediate electron carriers: a diheme cytochrome c 4 and a single heme cytochrome c 5 . Together, these proteins allow electron transfer from ubiquinol in cytochrome bc 1 to oxygen in cytochrome cbb 3 . We also present evidence that different isoforms of cytochrome cbb 3 can participate in formation of this SC without changing the overall SC architecture. Incorporating these different subunit isoforms into the SC would allow the bacterium to adapt to different environmental conditions. Bioinformatic analysis focusing on structural motifs in the SC suggests that cytochrome bc 1 - cbb 3 SCs also exist in other bacterial pathogens.

The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualise the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide-induced conformational changes that create a docking site for the substrate binding MIDAS domain on the AAA +ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.

Gyrase is a unique type IIA topoisomerase that uses ATP hydrolysis to maintain the negatively supercoiled state of bacterial DNA. In order to perform its function, gyrase undergoes a sequence of conformational changes that consist of concerted gate openings, DNA cleavage, and DNA strand passage events. Structures where the transported DNA molecule (T-segment) is trapped by the A subunit have not been observed. Here we present the cryoEM structures of two oligomeric complexes of open gyrase A dimers and DNA. The protein subunits in these complexes were solved to 4 Å and 5.2 Å resolution. One of the complexes traps a linear DNA molecule, a putative T-segment, which interacts with the open gyrase A dimers in two states, representing steps either prior to or after passage through the DNA-gate. The structures locate the T-segment in important intermediate conformations of the catalytic cycle and provide insights into gyrase-DNA interactions and mechanism.

VRC01 broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4BS) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env). Unlike mature antibodies, corresponding VRC01 germline precursors poorly bind to Env. Immunogen design has mostly relied on glycan removal from trimeric Env constructs and has had limited success in eliciting mature VRC01 bnAbs. To better understand elicitation of such bnAbs, we characterized the inferred germline precursor of VRC01 in complex with a modified trimeric 426c Env by cryo-electron microscopy and a 426c gp120 core by X-ray crystallography, biolayer interferometry, immunoprecipitation, and glycoproteomics. Our results show VRC01 germline antibodies interacted with a wild-type 426c core lacking variable loops 1–3 in the presence and absence of a glycan at position Asn276, with the latter form binding with higher affinity than the former. Interactions in the presence of an Asn276 oligosaccharide could be enhanced upon carbohydrate shortening, which should be considered for immunogen design.

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein VLP derived from DENV serotype-2 were engineered becoming highly matured (mD2VLP) and showed variable size distribution with diameter of ~31 nm forming the major population under cryo-electron microscopy examination. Furthermore, mD2VLP particles of 31 nm diameter possess a T = 1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Mice vaccinated with mD2VLP generated higher cross-reactive (CR) neutralization antibodies (NtAbs) and were fully protected against all 4 serotypes of DENV. Our results highlight the potential of ‘epitope-resurfaced’ mature-form D2VLPs in inducing quaternary structure-recognizing broad CR NtAbs to guide future dengue vaccine design.

Lewy bodies, hallmarks of Parkinson's disease, contain C-terminally truncated (ΔC) α-synuclein (α-syn). Here, we report fibril structures of three N-terminally acetylated (Ac) α-syn constructs, Ac1–140, Ac1–122, and Ac1–103, solved by cryoelectron microscopy. Both ΔC-α-syn variants exhibited faster aggregation kinetics, and Ac1–103 fibrils efficiently seeded the full-length protein, highlighting their importance in pathogenesis. Interestingly, fibril helical twists increased upon the removal of C-terminal residues and can be propagated through cross-seeding. Compared to that of Ac1–140, increased electron densities were seen in the N-terminus of Ac1–103, whereas the C-terminus of Ac1–122 appeared more structured. In accord, the respective termini of ΔC-α-syn exhibited increased protease resistance. Despite similar amyloid core residues, distinctive features were seen for both Ac1–122 and Ac1–103. Particularly, Ac1–103 has the tightest packed core with an additional turn, likely attributable to conformational changes in the N-terminal region. These molecular differences offer insights into the effect of C-terminal truncations on α-syn fibril polymorphism. Unlabelled Image • Effects of C-terminal truncations on α-syn fibril formation were studied. • Fibril helical twists increased upon the removal of C-terminal residues. • Fibril structures of full length and ΔC-α-syn were solved by cryoEM. • Conformational differences were revealed at N- and C-termini. • Structural insights were gained on α-syn fibril polymorphism. [ABSTRACT FROM AUTHOR]

Bluetongue virus (BTV), a major threat to livestock, is a multilayered, nonturreted member of the Reoviridae, a family of segmented dsRNA viruses characterized by endogenous RNA transcription through an RNA-dependent RNA polymerase (RdRp). To date, the structure of BTV RdRp has been unknown, limiting our mechanistic understanding of BTV transcription and hindering rational drug design effort targeting this essential enzyme. Here, we report the in situ structures of BTV RdRp VP1 in both the triple-layered virion and double-layered core, as determined by cryo-electron microscopy (cryoEM) and subparticle reconstruction. BTV RdRp has 2 unique motifs not found in other viral RdRps: a fingernail, attached to the conserved fingers subdomain, and a bundle of 3 helices: 1 from the palm subdomain and 2 from the N-terminal domain. BTV RdRp VP1 is anchored to the inner surface of the capsid shell via 5 asymmetrically arranged N termini of the inner capsid shell protein VP3A around the 5-fold axis. The structural changes of RdRp VP1 and associated capsid shell proteins between BTV virions and cores suggest that the detachment of the outer capsid proteins VP2 and VP5 during viral entry induces both global movements of the inner capsid shell and local conformational changes of the N-terminal latch helix (residues 34 to 51) of 1 inner capsid shell protein VP3A, priming RdRp VP1 within the capsid for transcription. Understanding this mechanism in BTV also provides general insights into RdRp activation and regulation during viral entry of other multilayered, nonturreted dsRNA viruses. [ABSTRACT FROM AUTHOR]

Highlights • Carbon elastic and inelastic electron scattering cross sections are measured vs. energy. • Elastic scattering is compared to energy deposition and radiation damage. • An optimal energy for cryoEM of a given biological specimen thickness is determined. Abstract We have measured the dependence on electron energy of elastic and inelastic scattering cross-sections from carbon, over the energy range that includes 100 keV to 300 keV. We also compared quantitatively the radiation damage to bacteriorhodopsin and paraffin (C 44 H 90) at 100 keV and 300 keV by observing the fading of the diffraction spots from two-dimensional crystals as a function of electron fluence. The elastic cross-section is 2.01 - fold greater at 100 keV than at 300 keV, whereas the radiation damage increased by only 1.57. This implies that the amount of useful information from diffraction patterns or images of most biological structures should be 25% greater using 100 keV rather than 300 keV electrons. Using these measurements, we calculate the energy dependence of the available information per unit damage for a specimen of a particular thickness, which we call the "information coefficient." This allows us to determine the optimal energy for imaging a biological specimen of a given thickness. We find that for most single particle cryoEM specimens, 100 keV provides not only the highest potential for information per unit damage, but would also simplify the instrument while retaining the potential to reach high resolution with a minimum of data. These measurements will help guide the development and use of electron cryomicroscopes for biology. [ABSTRACT FROM AUTHOR]

With recent technological advances, the atomic resolution structure of any purified biomolecular complex can, in principle, be determined by single-particle electron cryomicroscopy (cryoEM). In practice, the primary barrier to structure determination is the preparation of a frozen specimen suitable for high-resolution imaging. To address this, we present a multifunctional specimen support for cryoEM, comprising large-crystal monolayer graphene suspended across the surface of an ultrastable gold specimen support. Using a low-energy plasma surface modification system, we tune the surface of this support to the specimen by patterning a range of covalent functionalizations across the graphene layer on a single grid. This support design reduces specimen movement during imaging, improves image quality, and allows highresolution structure determination with a minimum of material and data. [ABSTRACT FROM AUTHOR]

• A procedure for automating TEM and electron detector to collect MicroED data is described. • The widely-used open-source software program SerialEM is used to coordinate TEM goniometer movement and detector image recording. • A SerialEM script and usage protocol for Thermo Fisher TEMs is provided. The cryoEM method Microcrystal Electron Diffraction (MicroED) involves transmission electron microscope (TEM) and electron detector working in synchrony to collect electron diffraction data by continuous rotation. We previously reported several protein, peptide, and small molecule structures by MicroED using manual control of the microscope and detector to collect data. Here we present a procedure to automate this process using a script developed for the popular open-source software package SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. Depending on crystal and substrate geometry, more than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses. [ABSTRACT FROM AUTHOR]

Abstract Membrane protein stabilization after detergent solubilization presents drawbacks for structural and biophysical studies, in particular that of a reduced stability in detergent micelles. Therefore, alternative methods are required for efficient stabilization. Lipid nanodisc made with the membrane scaffold protein MSP is a valuable system but requires a fine optimization of the lipid to protein ratio. We present here the use of the scaffold protein MSP without added lipids as a minimal system to stabilize membrane proteins. We show that this method is applicable to α-helical and β-strands transmembrane proteins. This method allowed cryo-electron microscopy structural study of the bacterial transporter MexB. A protein quantification indicates that MexB is stabilized by two MSP proteins. This simplified and efficient method proposes a new advance in harnessing the MSP potential to stabilize membrane proteins. Graphical abstract Unlabelled Image Highlights • Scaffold protein MSP without added lipids stabilizes membrane proteins. • Minimal MSP nanodisc applicable to α-helical and β-strands transmembrane proteins • Minimal MSP nanodisc amenable to single particle cryoEM • Bacterial transporter MexB is stabilized by two MSP proteins. [ABSTRACT FROM AUTHOR]

Abstract Human plasma butyrylcholinesterase (BChE) is an endogenous bioscavenger that hydrolyzes numerous medicamentous and poisonous esters and scavenges potent organophosphorus nerve agents. BChE is thus a marker for the diagnosis of OP poisoning. It is also considered a therapeutic target against Alzheimer's disease. Although the X-ray structure of a partially deglycosylated monomer of human BChE was solved 15 years ago, all attempts to determine the 3D structure of the natural full-length glycosylated tetrameric human BChE have been unsuccessful so far. Here, a combination of three complementary structural methods—single-particle cryo-electron microscopy, molecular dynamics and small-angle X-ray scattering—were implemented to elucidate the overall structural and spatial organization of the natural tetrameric human plasma BChE. A 7.6 Å cryoEM map clearly shows the major features of the enzyme: a dimer of dimers with a nonplanar monomer arrangement, in which the interconnecting super helix complex PRAD-(WAT) 4 -peptide C-terminal tail is located in the center of the tetramer, nearly perpendicular to its plane, and is plunged deep between the four subunits. Molecular dynamics simulations allowed optimization of the geometry of the molecule and reconstruction of the structural features invisible in the cryoEM density, i.e., glycan chains and glycan interdimer contact areas, as well as intermonomer disulfide bridges at the C-terminal tail. Finally, SAXS data were used to confirm the consistency of the obtained model with the experimental data. The tetramer organization of BChE is unique in that the four subunits are joined at their C-termini through noncovalent contacts with a short polyproline-rich peptide. This tetramer structure could serve as a model for the design of highly stable glycosylated tetramers. Graphical abstract Image 1 Highlights • The 3D structure of natural human BChE tetramer was elucidated by cryoEM at a resolution of 7.6 Å. • Nonflat subunit organization with a PRAD-C-terminal bundle oriented vertically and plunged deep between the 4 subunits. • All-atom MD simulations to improve the model show glycan chains and disulfide bonds at the C-terminal tail. • SAXS experiments were conducted to check the consistency of the obtained model of BChE tetramer. [ABSTRACT FROM AUTHOR]

Abstract With the rapidly growing number of macromolecular structures solved to near-atomic resolution using electron cryomicroscopy (cryoEM), map interpretation and model building directly from the density without the use of structural templates has become increasingly important. As part of the 2015/2016 Map and Model Challenge, we attempted to assess our latest de novo modeling tool, Pathwalking, in terms of performance and usability, as well as identify areas for future improvements. In total, we applied Pathwalking to six density maps between 3 and 4.5 Å resolution selected from the challenge data sets. In five of the six cases, Pathwalking was able to accurately determine the protein fold and in three of these cases, the final all atom model had less than 1.6 Å RMSD when compared to the known structure. Model building and refinement was nearly completely automated, used default parameters and took less than 30 min to complete a refined all atom model. A direct outgrowth of this work was a more streamlined automated command line Pathwalking utility, as well as a novel sequence assignment and optimization routine, which attempts to register sidechain density with expected side chain volume. In total, Pathwalking offers a nearly complete, robust and efficient method for constructing atomistic protein structures directly from a density map without the aid of a template. [ABSTRACT FROM AUTHOR]

Highlights • Release of EMAN2.21, with changes inspired by the Map Challenge. • High-resolution SPR maps are now filtered to enhance sidechain visibility. • A new neural network-based particle picker provides accurate picking even on challenging data. • Per-particle CTF correction routine now fully automated. • Development of a new algorithm for identifying "bad" particles. Abstract EMAN2 is an extensible software suite with complete workflows for performing high-resolution single particle analysis, 2-D and 3-D heterogeneity analysis, and subtomogram averaging, among other tasks. Participation in the recent CryoEM Map Challenge sponsored by the EMDatabank led to a number of significant improvements to the single particle analysis process in EMAN2. A new convolutional neural network particle picker was developed, which dramatically improves particle picking accuracy for difficult data sets. A new particle quality metric capable of accurately identifying "bad" particles with a high degree of accuracy, no human input, and a negligible amount of additional computation, has been introduced, and this now serves as a replacement for earlier human-biased methods. The way 3-D single particle reconstructions are filtered has been altered to be more comparable to the filter applied in several other popular software packages, dramatically improving the appearance of sidechains in high-resolution structures. Finally, an option has been added to perform local resolution-based iterative filtration, resulting in local resolution improvements in many maps. [ABSTRACT FROM AUTHOR]

BAM, Cryoem, Darobactin, In-Cell EPR, PELDOR Keywords: BAM; Cryoem; Darobactin; In-Cell EPR; PELDOR EN BAM Cryoem Darobactin In-Cell EPR PELDOR 1 1 1 08/21/23 20230821 NES 230821 B The I i -barrel assembly machine b (BAM) is essential for folding outer membrane proteins (OMPs) into the outer membrane (OM) of Gram-negative bacteria. Inside Cover: Darobactin B Stabilises a Lateral-Closed Conformation of the BAM Complex in E. coli Cells (Angew. [Extracted from the article]

Keywords: BAM; Cryoem; Darobactin; In-Cell EPR; PELDOR EN BAM Cryoem Darobactin In-Cell EPR PELDOR 1 1 1 08/17/23 20230821 NES 230821 B The I i -barrel assembly machine b (BAM) is essential for folding outer membrane proteins (OMPs) into the outer membrane (OM) of Gram-negative bacteria. Structures of BAM have been solved using X-ray crystallography and cryoEM, but the conformation of BAM in the highly crowded native OM remained unsolved. BAM, Cryoem, Darobactin, In-Cell EPR, PELDOR. [Extracted from the article]

In this overview, we briefly outline recent advances in electron cryomicroscopy (cryoEM) and explain why the journal IUCrJ, published by the International Union of Crystallography, could provide a natural home for publications covering many present and future developments in the cryoEM field.

Here, we describe the structure of three actinobacteriophage capsids that infect Mycobacterium smegmatis. The capsid structures were resolved to approximately six angstroms, which allowed confirmation that each bacteriophage uses the HK97-fold to form their capsid. One bacteriophage, Rosebush, may have a novel variation of the HK97-fold. Four novel accessory proteins that form the capsid head along with the major capsid protein were identified. Two of the accessory proteins were minor capsid proteins and showed some homology, based on bioinformatic analysis, to the TW1 bacteriophage. The remaining two accessory proteins are decoration proteins that are located on the outside of the capsid and do not resemble any previously described bacteriophage decoration protein. SDS-PAGE and mass spectrometry was used to identify the accessory proteins and bioinformatic analysis of the accessory proteins suggest they are used in many actinobacteriophage capsids.

The nuclear RNA exosome complex mediates the processing of structured RNAs and the decay of aberrant non-coding RNAs, an important function particularly in human cells. Most mechanistic studies to date have focused on the yeast system. Here, we reconstituted and studied the properties of a recombinant 14-subunit human nuclear exosome complex. In biochemical assays, the human exosome embeds a longer RNA channel than its yeast counterpart. The 3.8 Å resolution cryo-EM structure of the core complex bound to a single-stranded RNA reveals that the RNA channel path is formed by two distinct features of the hDIS3 exoribonuclease: an open conformation and a domain organization more similar to bacterial RNase II than to yeast Rrp44. The cryo-EM structure of the holo-complex shows how obligate nuclear cofactors position the hMTR4 helicase at the entrance of the core complex, suggesting a striking structural conservation from lower to higher eukaryotes.

Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies.

Near atomic-resolution structures have provided insights into the mechanisms by which the Piezo1 ion channel senses and responds to mechanical stimuli.

Mechanosensitive ion channels convert external mechanical stimuli into electrochemical signals for critical processes including touch sensation, balance, and cardiovascular regulation. The best understood mechanosensitive channel, MscL, opens a wide pore, which accounts for mechanosensitive gating due to in-plane area expansion. Eukaryotic Piezo channels have a narrow pore and therefore must capture mechanical forces to control gating in another way. We present a cryo-EM structure of mouse Piezo1 in a closed conformation at 3.7Å-resolution. The channel is a triskelion with arms consisting of repeated arrays of 4-TM structural units surrounding a pore. Its shape deforms the membrane locally into a dome. We present a hypothesis in which the membrane deformation changes upon channel opening. Quantitatively, membrane tension will alter gating energetics in proportion to the change in projected area under the dome. This mechanism can account for highly sensitive mechanical gating in the setting of a narrow, cation-selective pore.

First discovered in bacteriophage HK97, biological chainmail is a highly stable system formed by concatenated protein rings. Each subunit of the ring contains the HK97-like fold, which is characterized by its submarine-like shape with a 5-stranded β sheet in the axial (A) domain, spine helix in the peripheral (P) domain, and an extended (E) loop. HK97 capsid consists of covalently-linked copies of just one HK97-like fold protein and represents the most effective strategy to form highly stable chainmail needed for dsDNA genome encapsidation. Recently, near-atomic resolution structures enabled by cryo electron microscopy (cryoEM) have revealed a range of other, more complex variants of this strategy for constructing dsDNA viruses. The first strategy, exemplified by P22-like phages, is the attachment of an insertional (I) domain to the core 5-stranded β sheet of the HK97-like fold. The atomic models of the Bordetella phage BPP-1 showcases an alternative topology of the classic HK97 topology of the HK97-like fold, as well as the second strategy for constructing stable capsids, where an auxiliary jellyroll protein dimer serves to cement the non-covalent chainmail formed by capsid protein subunits. The third strategy, found in lambda-like phages, uses auxiliary protein trimers to stabilize the underlying non-covalent chainmail near the 3-fold axis. Herpesviruses represent highly complex viruses that use a combination of these strategies, resulting in four-level hierarchical organization including a non-covalent chainmail formed by the HK97-like fold domain found in the floor region. A thorough understanding of these structures should help unlock the enigma of the emergence and evolution of dsDNA viruses and inform bioengineering efforts based on these viruses.

Structural biology continues to benefit from an expanding toolkit, which is helping to gain unprecedented insight into the assembly and organization of multi-protein machineries, enzyme mechanisms and ligand/inhibitor binding. During the last ten years, cryoEM has become widely available and has provided a major boost to structure determination of membrane proteins and large multi-protein complexes. Many of the structures have now been made available at resolutions around 2 Å, where fundamental questions regarding enzyme mechanisms can be addressed. Over the years, the abbreviation cryoEM has been understood to stand for different things. We wish the wider community to engage and clarify the definition of cryoEM so that the expanding literature involving cryoEM is unified., (open access.)

The great success of single-particle electron cryo-microscopy (cryoEM) during the last decade has involved the development of powerful new computer programs and packages that guide the user along a recommended processing workflow, in which the wisdom and choices made by the developers help everyone, especially new users, to obtain excellent results. The ability to carry out novel, non-standard or unusual combinations of image-processing steps is sometimes compromised by the convenience of a standard procedure. Some of the older programs were written with great flexibility and are still very valuable. Among these, the original MRC image-processing programs for structure determination by 2D crystal and helical processing alongside general-purpose utility programs such as Ximdisp, label, imedit and twofile are still available. This work describes an updated version of the MRC software package (MRC2020) that is freely available from CCP-EM. It includes new features and improvements such as extensions to the MRC format that retain the versatility of the package and make it particularly useful for testing novel computational procedures in cryoEM., (open access.)

Invasive aspergillosis is one of the most serious clinical invasive fungal infections, resulting in a high case fatality rate among immunocompromised patients. The disease is caused by saprophytic molds in the genus Aspergillus , including Aspergillus fumigatus , the most significant pathogenic species. The fungal cell wall, an essential structure mainly composed of glucan, chitin, galactomannan, and galactosaminogalactan, represents an important target for the development of antifungal drugs. UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) is a central enzyme in the metabolism of carbohydrates that catalyzes the biosynthesis of UDP-glucose, a key precursor of fungal cell wall polysaccharides. Here, we demonstrate that the function of UGP is vital for Aspergillus nidulans ( An UGP). To understand the molecular basis of An UGP function, we describe a cryoEM structure (global resolution of 3.5 Å for the locally refined subunit and 4 Å for the octameric complex) of a native An UGP. The structure reveals an octameric architecture with each subunit comprising an N-terminal α-helical domain, a central catalytic glycosyltransferase A-like (GT-A-like) domain, and a C-terminal (CT) left-handed β-helix oligomerization domain. An UGP displays unprecedented conformational variability between the CT oligomerization domain and the central GT-A-like catalytic domain. In combination with activity measurements and bioinformatics analysis, we unveil the molecular mechanism of substrate recognition and specificity for An UGP. Altogether, our study not only contributes to understanding the molecular mechanism of catalysis/regulation of an important class of enzymes but also provides the genetic, biochemical, and structural groundwork for the future exploitation of UGP as a potential antifungal target. IMPORTANCE Fungi cause diverse diseases in humans, ranging from allergic syndromes to life-threatening invasive diseases, together affecting more than a billion people worldwide. Increasing drug resistance in Aspergillus species represents an emerging global health threat, making the design of antifungals with novel mechanisms of action a worldwide priority. The cryoEM structure of UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) from the filamentous fungus Aspergillus nidulans reveals an octameric architecture displaying unprecedented conformational variability between the C-terminal oligomerization domain and the central glycosyltransferase A-like catalytic domain in the individual protomers. While the active site and oligomerization interfaces are more highly conserved, these dynamic interfaces include motifs restricted to specific clades of filamentous fungi. Functional study of these motifs could lead to the definition of new targets for antifungals inhibiting UGP activity and, thus, the architecture of the cell wall of filamentous fungal pathogens., Competing Interests: The authors declare no conflict of interest.

As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids. However, the reasons for such misbehavior are not well understood, which limits systematic approaches to solving the problem. Here, we have developed a theoretical formulation that explains these observations. Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy-rationalizing the use of surfactant, which is a direct solution to reduce the surface tension of the aqueous solution. By performing cryogenic electron tomography (cryoET) on the widely-tested sample, GroEL, we demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradually reducing the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that suitable surfactants do not damage the biological complex, thus suggesting that they might provide a practical, simple, and general solution to the problem for high-resolution cryoEM. Applying this solution to a real-world AWI adsorption problem involving a more challenging membrane protein, namely, the ClC-1 channel, has resulted in its near-atomic structure determination using cryoEM., Competing Interests: Joon S. Kang, Xueting Zhou, Yun-Tao Liu, Kaituo Wang and Z. Hong Zhou declare that they have no conflict of interest., (© The Author(s) 2023.)

The TOM complex is the main entry point for precursor proteins (preproteins) into mitochondria. Preproteins containing targeting sequences are recognized by the TOM complex and imported into mitochondria. We have determined the structure of the TOM core complex from Neurospora crassa by single-particle electron cryomicroscopy at 3.3 Å resolution, showing its interaction with a bound preprotein at 4 Å resolution, and of the TOM holo complex including the Tom20 receptor at 6 to 7 Å resolution. TOM is a transmembrane complex consisting of two β-barrels, three receptor subunits, and three short transmembrane subunits. Tom20 has a transmembrane helix and a receptor domain on the cytoplasmic side. We propose that Tom20 acts as a dynamic gatekeeper, guiding preproteins into the pores of the TOM complex. We analyze the interactions of Tom20 with other TOM subunits, present insights into the structure of the TOM holo complex, and suggest a translocation mechanism.

The β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH.)