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Aims This study assessed the inter-rater reliability, acceptability and usability of the Medication Administration Evaluation and Feedback Tool for nurses in the clinical setting. Background Medication administration is a complex nursing task requiring multiple steps to ensure safe and accurate delivery of medications to patients. Currently, registered nurses are not routinely provided the opportunity for regular review of their practice. The Medication Administration Evaluation and Feedback Tool has been previously validated in the simulated environment. Methods Four nurse observers were trained to use the tool. Thirty nurses participated to be observed in the clinical setting. Each nurse was assessed simultaneously by two observers. Inter-rater reliability was assessed using Fleiss’ Kappa coefficient. A postobservation survey was conducted to assess user acceptability. The Guideline for Reporting Reliability and Agreement Studies Enhancing the Quality and Transparency of Health Research was used. Results The observed agreement between observers using the Medication Administration Evaluation and Feedback Tool in clinical practice was 0.90 and Fleiss’ kappa coefficient was 0.77 demonstrating excellent agreement and inter-rater reliability. Both nurses and observers reported the tool was useful and practical for use in evaluating medication administration practice in the clinical environment. Conclusions Inter-rater reliability testing of the Medication Administration Evaluation and Feedback Tool in the clinical environment demonstrated it is a reliable and valid tool when used by different observers. Both nurses and observers found using the tool a positive and useful experience when evaluating medication administration practice.

Background Peripheral intravenous catheters (PIVCs) are ubiquitous medical devices, crucial to providing essential fluids and drugs. However, post-insertion PIVC failure occurs frequently, likely due to inconsistent maintenance practice such as flushing. The aim of this implementation study was to evaluate the impact a multifaceted intervention centred on short PIVC maintenance had on patient outcomes. Methods This single-centre, incomplete, stepped wedge, cluster randomised trial with an implementation period was undertaken at a quaternary hospital in Queensland, Australia. Eligible patients were from general medical and surgical wards, aged ≥ 18 years, and requiring a PIVC for > 24 h. Wards were the unit of randomisation and allocation was concealed until the time of crossover to the implementation phase. Patients, clinicians, and researchers were not masked but infections were adjudicated by a physician masked to allocation. Practice during the control period was standard care (variable practice with manually prepared flushes of 0.9% sodium chloride). The intervention group received education reinforcing practice guidelines (including administration with manufacturer-prepared pre-filled flush syringes). The primary outcome was all-cause PIVC failure (as a composite of occlusion, infiltration, dislodgement, phlebitis, and primary bloodstream or local infection). Analysis was by intention-to-treat. Results Between July 2016 and February 2017, 619 patients from 9 clusters (wards) were enrolled (control n = 306, intervention n = 313), with 617 patients comprising the intention-to-treat population. PIVC failure was 91 (30%) in the control and 69 (22%) in the intervention group (risk difference − 8%, 95% CI − 14 to − 1, p = 0.032). Total costs were lower in the intervention group. No serious adverse events related to study intervention occurred. Conclusions This study demonstrated the effectiveness of post-insertion PIVC flushing according to recommended guidelines. Evidence-based education, surveillance and products for post-insertion PIVC management are vital to improve patient outcomes. Trial registration Trial submitted for registration on 25 January 2016. Approved and retrospectively registered on 4 August 2016. Ref: ACTRN12616001035415.

Cells of most bacterial species are around 2 µm in length, with some of the largest specimens reaching 750 µm. Using fluorescence, x-ray, and electron microscopy in conjunction with genome sequencing, we characterized Ca. Thiomargarita magnifica, a bacterium with an average cell length greater than 9,000 µm that is visible to the naked eye. We found that these cells grow orders of magnitude over theoretical limits for bacterial cell size through unique biology, display unprecedented polyploidy of more than half a million copies of a very large genome, and undergo a dimorphic life cycle with asymmetric segregation of chromosomes in daughter cells. These features, along with compartmentalization of genomic material and protein synthesis in membrane-bound organelles, indicate gain of complexity in the Thiomargarita lineage, and challenge traditional concepts of bacterial cells.One Sentence SummaryCa. T. magnifica are compartmentalized centimeter-long bacteria

SummaryLipid A, the membrane-anchored portion of lipopolysaccharide, is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here, we identify regulatory and structural factors that together permit Caulobacter crescentus to eliminate lipid A from its OM. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog which we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to eliminate lipid A. Fitness of lipid A-deficient Caulobacter requires a previously uncharacterized anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.

Actin assembly provides force for a multitude of cellular processes. Compared to actin-assembly-based force production during cell migration, relatively little is understood about how actin assembly generates pulling forces for vesicle formation. Here, cryo-electron tomography identified actin filament number, organization, and orientation during clathrin-mediated endocytosis in human SK-MEL-2 cells, showing that force generation is robust despite variance in network organization. Actin dynamics simulations incorporating ameasured branch angle indicate that sufficient force to drive membrane internalization is generated throughpolymerization and that assembly is triggered from ∼4 founding "mother" filaments, consistent with tomography data. Hip1R actin filament anchoring points are present along the entire endocytic invagination, where simulations show that it is key to pulling force generation, and along the neck, where it targets filament growth and makes internalization more robust. Actin organization described here allowed direct translation of structure to mechanism with broad implications for other actin-driven processes.

SummaryActin assembly provides force for a multitude of cellular processes. Compared to actin assembly- based force production during cell migration, relatively little is understood about how actin assembly generates pulling forces for vesicle formation. Here, cryo-electron tomography revealed actin filament number, organization, and orientation during clathrin-mediated endocytosis in human cells, showing that force generation is robust despite variance in network organization. Actin dynamics simulations incorporating a measured branch angle indicate that sufficient force to drive membrane internalization is generated through polymerization, and that assembly is triggered from ∼4 founding “mother” filaments, consistent with tomography data. Hip1R actin filament anchoring points are present along the entire endocytic invagination, where simulations show that it is key to pulling force generation, and along the neck, where it targets filament growth and makes internalization more robust. Actin cytoskeleton organization described here allowed direct translation of structure to mechanism with broad implications for other actin-driven processes.Highlights-Filament anchorage points are key to pulling force generation and efficiency.-Native state description of CME-associated actin force-producing networks.-Branched actin filament assembly is triggered from multiple mother filaments.-Actin force production is robust despite considerable network variability.

Carboxysomes are capsid-like, CO2-fixing organelles that are present in all cyanobacteria and some chemoautotrophs and that substantially contribute to global primary production. They are composed of a selectively permeable protein shell that encapsulates Rubisco, the principal CO2-fixing enzyme, and carbonic anhydrase. As the centerpiece of the carbon-concentrating mechanism, by packaging enzymes that collectively enhance catalysis, the carboxysome shell enables the generation of a locally elevated concentration of substrate CO2 and the prevention of CO2 escape. A functional carboxysome consisting of an intact shell and cargo is essential for cyanobacterial growth under ambient CO2 concentrations. Using cryo-electron microscopy, we have determined the structure of a recombinantly produced simplified β-carboxysome shell. The structure reveals the sidedness and the specific interactions between the carboxysome shell proteins. The model provides insight into the structural basis of selective permeability of the carboxysome shell and can be used to design modifications to investigate the mechanisms of cargo encapsulation and other physiochemical properties such as permeability. Notably, the permeability properties are of great interest for modeling and evaluating this carbon-concentrating mechanism in metabolic engineering. Moreover, we find striking similarity between the carboxysome shell and the structurally characterized, evolutionarily distant metabolosome shell, implying universal architectural principles for bacterial microcompartment shells.

Background Incorrect medication administration risks errors and patient harm. The aim of this study was to test the reliability of the newly developed medication administration evaluation and feedback tool. Methods An observational, fully crossed design using recorded scenarios in a simulated environment was used to test reliability and agreement of an evaluation tool for nurses administering medications. Results Intrarater and inter-rater reliability observed agreement overall were 84% and 82% with Fleiss Kappa coefficient 0.72 and 0.68, respectively. Overall intrarater and inter-rater reliability of the tool rated as good. Conclusions The medication administration evaluation and feedback tool is reliable in a simulated environment.

Cryo-electron tomography (cryo-ET) is an extremely powerful tool which is used to image cellular features in their close-to-native environment at a resolution where both protein structure and membrane morphology can be revealed. Compared to conventional electron microscopy methods for biology, cryo-ET does not include the use of potentially artifact generating agents for sample fixation or visualization. Despite its obvious advantages, cryo-ET has not been widely adopted by cell biologists. This might originate from the overwhelming and constantly growing number of complex ways to record and process data as well as the numerous methods available for sample preparation. In this chapter, we will take one step back and guide the reader through the essential steps of sample preparation using mammalian cells, as well as the basic steps involved in data recording and processing. The described protocol will allow the reader to obtain data that can be used for morphological analysis and precise measurements of biological structures in their cellular environment. Furthermore, this data can be used for more elaborate structural analysis by applying further image processing steps like subtomogram averaging, which is required to determine the structure of proteins.

Lipid A, the membrane-anchored portion of lipopolysaccharide (LPS), is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here we identify regulatory and structural factors that together render lipid A nonessential in Caulobacter crescentus. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to survive when lipid A synthesis is blocked. Fitness of lipid A-deficient Caulobacter requires an anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.

We describe how to use several machine learning techniques organized in a learning pipeline to segment and identify subcellular structures from cryo electron tomograms. These tomograms are difficult to analyze with traditional segmentation tools. The learning pipeline in our approach starts from supervised learning via a special convolutional neural network trained with simulated data. It continues with semi-supervised reinforcement learning and/or a region merging techniques that try to piece together disconnected components that should belong to the same subcellular structure. A parametric or non-parametric fitting procedure is then used to enhance the segmentation results and quantify uncertainties in the fitting. Domain knowledge is used in generating the training data for the neural network and in guiding the fitting procedure through the use of appropriately chosen priors and constraints. We demonstrate that the approach proposed here work well for extracting membrane surfaces of protein reconstituted liposomes in a cellular environment that contains other artifacts.

Rows of ATP synthase dimers define the cristae morphology of the inner mitochondrial membrane. Anselmi et al. use molecular simulations to show that the formation of these rows is spontaneous and driven by an attractive force induced by the membrane, not direct protein–protein interactions., Adenosine triphosphate (ATP) synthases populate the inner membranes of mitochondria, where they produce the majority of the ATP required by the cell. From yeast to vertebrates, cryoelectron tomograms of these membranes have consistently revealed a very precise organization of these enzymes. Rather than being scattered throughout the membrane, the ATP synthases form dimers, and these dimers are organized into rows that extend for hundreds of nanometers. The rows are only observed in the membrane invaginations known as cristae, specifically along their sharply curved edges. Although the presence of these macromolecular structures has been irrefutably linked to the proper development of cristae morphology, it has been unclear what drives the formation of the rows and why they are specifically localized in the cristae. In this study, we present a quantitative molecular-simulation analysis that strongly suggests that the dimers of ATP synthases organize into rows spontaneously, driven by a long-range attractive force that arises from the relief of the overall elastic strain of the membrane. The strain is caused by the V-like shape of the dimers, unique among membrane protein complexes, which induces a strong deformation in the surrounding membrane. The process of row formation is therefore not a result of direct protein–protein interactions or a specific lipid composition of the membrane. We further hypothesize that, once assembled, the ATP synthase dimer rows prime the inner mitochondrial membrane to develop folds and invaginations by causing macroscopic membrane ridges that ultimately become the edges of cristae. In this way, mitochondrial ATP synthases would contribute to the generation of a morphology that maximizes the surface area of the inner membrane, and thus ATP production. Finally, we outline key experiments that would be required to verify or refute this hypothesis.

The inner boundary and the cristae membrane are connected by pore-like structures termed crista junctions (CJs). The MICOS complex is required for CJ formation and enriched at CJs. Here, we address the roles of the MICOS subunits Mic27 and Mic10. We observe a positive genetic interaction be-tween Mic27 and Mic60 and deletion of Mic27 results in impaired formation of CJs and altered cristae membrane curvature. Mic27 acts in an antagonistic manner to Mic60 as it promotes oligomerization of the F1FO-ATP synthase and partially restores CJ formation in cells lacking Mic60. Mic10 impairs oli-gomerization of the F1FO-ATP synthase similar to Mic60. Applying complex-ome profiling, we observed that deletion of Mic27 destabilizes the MICOS complex but does not impair formation of a high molecular weight Mic10 subcomplex. Moreover, this Mic10 subcomplex comigrates with the dimeric F1FO-ATP synthase in a Mic27-independent manner. Further, we observed a chemical crosslink of Mic10 to Mic27 and of Mic10 to the F1FO-ATP synthase subunit e. We corroborate the physical interaction of the MICOS complex and the F1FO-ATP synthase. We propose a model in which part of the F1FO-ATP synthase is linked to the MICOS complex via Mic10 and Mic27 and by that is regulating CJ formation.

Carboxysomes are capsid-like, CO(2)-fixing organelles that are present in all cyanobacteria and some chemoautotrophs and that substantially contribute to global primary production. They are composed of a selectively permeable protein shell that encapsulates Rubisco, the principal CO(2)-fixing enzyme, and carbonic anhydrase. As the centerpiece of the carbon-concentrating mechanism, by packaging enzymes that collectively enhance catalysis, the carboxysome shell enables the generation of a locally elevated concentration of substrate CO(2) and the prevention of CO(2) escape. A functional carboxysome consisting of an intact shell and cargo is essential for cyanobacterial growth under ambient CO(2) concentrations. Using cryo-electron microscopy, we have determined the structure of a recombinantly produced simplified β-carboxysome shell. The structure reveals the sidedness and the specific interactions between the carboxysome shell proteins. The model provides insight into the structural basis of selective permeability of the carboxysome shell and can be used to design modifications to investigate the mechanisms of cargo encapsulation and other physiochemical properties such as permeability. Notably, the permeability properties are of great interest for modeling and evaluating this carbon-concentrating mechanism in metabolic engineering. Moreover, we find striking similarity between the carboxysome shell and the structurally characterized, evolutionarily distant metabolosome shell, implying universal architectural principles for bacterial microcompartment shells.

Significance The ATP synthase in the inner membrane of mitochondria generates most of the ATP that enables higher organisms to live. The inner membrane forms deep invaginations called cristae. Mitochondrial ATP synthases are dimeric complexes of two identical monomers. It is known that the ATP synthase dimers form rows along the tightly curved cristae ridges. Computer simulations suggest that the dimer rows bend the membrane locally, but this has not been shown experimentally. In this study, we use electron cryotomography to provide experimental proof that ATP synthase dimers assemble spontaneously into rows upon membrane reconstitution, and that these rows bend the membrane. The assembly of ATP synthase dimers into rows is most likely the first step in the formation of mitochondrial cristae., Mitochondrial ATP synthases form dimers, which assemble into long ribbons at the rims of the inner membrane cristae. We reconstituted detergent-purified mitochondrial ATP synthase dimers from the green algae Polytomella sp. and the yeast Yarrowia lipolytica into liposomes and examined them by electron cryotomography. Tomographic volumes revealed that ATP synthase dimers from both species self-assemble into rows and bend the lipid bilayer locally. The dimer rows and the induced degree of membrane curvature closely resemble those in the inner membrane cristae. Monomers of mitochondrial ATP synthase reconstituted into liposomes do not bend membrane visibly and do not form rows. No specific lipids or proteins other than ATP synthase dimers are required for row formation and membrane remodelling. Long rows of ATP synthase dimers are a conserved feature of mitochondrial inner membranes. They are required for cristae formation and a main factor in mitochondrial morphogenesis.

Photosynthetic NADH dehydrogenase-like complex type-1 (a.k.a, NDH, NDH-1, or NDH-1L) is a multi-subunit, membrane-bound oxidoreductase related to the respiratory complex I. Although originally discovered 30 years ago, a number of recent advances have revealed significant insight into the structure, function, and physiology of NDH-1. Here, we highlight progress in understanding the function of NDH-1 in the photosynthetic light reactions of both cyanobacteria and chloroplasts from biochemical and structural perspectives. We further examine the cyanobacterial-specific forms of NDH-1 that possess vectorial carbonic anhydrase (vCA) activity and function in the CO2-concentrating mechanism (CCM). We compare the proposed mechanism for the cyanobacterial NDH-1 vCA-activity to that of the DAB (DABs accumulates bicarbonate) complex, another putative vCA. Finally, we discuss both new and remaining questions pertaining to the mechanisms of NDH-1 complexes in light of these recent advances.

Cyclic electron flow around photosystem I (PSI) is a mechanism by which photosynthetic organisms balance the levels of ATP and NADPH necessary for efficient photosynthesis1,2. NAD(P)H dehydrogenase-like complex (NDH) is a key component of this pathway in most oxygenic photosynthetic organisms3,4 and is the last large photosynthetic membrane-protein complex for which the structure remains unknown. Related to the respiratory NADH dehydrogenase complex (complex I), NDH transfers electrons originating from PSI to the plastoquinone pool while pumping protons across the thylakoid membrane, thereby increasing the amount of ATP produced per NADP+ molecule reduced4,5. NDH possesses 11 of the 14 core complex I subunits, as well as several oxygenic-photosynthesis-specific (OPS) subunits that are conserved from cyanobacteria to plants3,6. However, the three core complex I subunits that are involved in accepting electrons from NAD(P)H are notably absent in NDH3,5,6, and it is therefore not clear how NDH acquires and transfers electrons to plastoquinone. It is proposed that the OPS subunits—specifically NdhS—enable NDH to accept electrons from its electron donor, ferredoxin3–5,7. Here we report a 3.1 A structure of the 0.42-MDa NDH complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, obtained by single-particle cryo-electron microscopy. Our maps reveal the structure and arrangement of the principal OPS subunits in the NDH complex, as well as an unexpected cofactor close to the plastoquinone-binding site in the peripheral arm. The location of the OPS subunits supports a role in electron transfer and defines two potential ferredoxin-binding sites at the apex of the peripheral arm. These results suggest that NDH could possess several electron transfer routes, which would serve to maximize plastoquinone reduction and avoid deleterious off-target chemistry of the semi-plastoquinone radical. The structure of NDH, a photosynthetic membrane-protein complex that is related to respiratory complex I, is obtained by single-particle cryo-electron microscopy.

Mitochondria generate the cellular fuel ATP to sustain complex life. Production of ATP depends on the oxidation of energy-rich compounds to produce the proton motive force (pmf), a chemical potential difference for protons, across the inner membrane. The pmf drives the ATP synthase to synthesize ATP via a mechanical rotary mechanism. The structures and functions of the protein components of this molecular machine, especially those involved directly in the catalytic formation of ATP, are widely conserved in metazoans, fungi, and eubacteria. Here we show that the proposal that this conservation does not extend to the ATP synthase from Trypanosoma brucei, a member of the euglenozoa and the causative agent of sleeping sickness in humans, is incorrect.

ATP synthases populate the inner membranes of mitochondria, where they produce the majority of the ATP required by the cell. Cryo-electron tomograms of these membranes from yeast to vertebrates have consistently revealed a very precise organization of these enzymes. Rather than being scattered throughout the membrane, the ATP synthases form dimers, and these dimers are organized into rows that extend for hundreds of nanometers. These rows are only observed in the membrane invaginations known as cristae, specifically along their sharply curved edges. Although the presence of these macromolecular structures has been irrefutably linked to the proper development of cristae morphology, it has been unclear what drives the formation of the rows and why they are specifically localized in the cristae. We present the result of a quantitative molecular-simulation analysis that strongly suggests that the ATP synthase dimers organize into rows spontaneously, driven by a long-ranged attractive force that results from relief in the overall elastic strain of the membrane. This strain is caused by the V-like shape of the dimers, unique among membrane-protein complexes, which induces a strong deformation in the surrounding membrane. The process of row formation is therefore not a result of protein-protein interactions, or of a specific lipid composition of the membrane. We further hypothesize that once assembled, the ATP synthase dimer rows prime the inner mitochondrial membrane to develop folds and invaginations, by causing macroscopic membrane ridges that ultimately become the cristae edges. In this view, mitochondrial ATP synthases would contribute to the generation of a morphology that maximizes the surface area of the inner membrane, and thus ATP production. Finally, we outline the key experiments that would be required to verify or refute this hypothesis.

Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.

ATP, the universal energy currency of cells, is produced by F-type ATP synthases, which are ancient, membrane-bound nanomachines. F-type ATP synthases use the energy of a transmembrane electrochemical gradient to generate ATP by rotary catalysis. Protons moving across the membrane drive a rotor ring composed of 8-15 c-subunits. A central stalk transmits the rotation of the c-ring to the catalytic F1 head, where a series of conformational changes results in ATP synthesis. A key unresolved question in this fundamental process is how protons pass through the membrane to drive ATP production. Mitochondrial ATP synthases form V-shaped homodimers in cristae membranes. Here we report the structure of a native and active mitochondrial ATP synthase dimer, determined by single-particle electron cryomicroscopy at 6.2 Å resolution. Our structure shows four long, horizontal membrane-intrinsic α-helices in the a-subunit, arranged in two hairpins at an angle of approximately 70° relative to the c-ring helices. It has been proposed that a strictly conserved membrane-embedded arginine in the a-subunit couples proton translocation to c-ring rotation. A fit of the conserved carboxy-terminal a-subunit sequence places the conserved arginine next to a proton-binding c-subunit glutamate. The map shows a slanting solvent-accessible channel that extends from the mitochondrial matrix to the conserved arginine. Another hydrophilic cavity on the lumenal membrane surface defines a direct route for the protons to an essential histidine-glutamate pair. Our results provide unique new insights into the structure and function of rotary ATP synthases and explain how ATP production is coupled to proton translocation.

We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis.

The natural cytotoxicity receptors, comprised of three type I membrane proteins NKp30, NKp44, and NKp46, are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. Among these, NKp30 is a major receptor targeting virus-infected cells, malignantly transformed cells, and immature dendritic cells. To date, only few cellular ligands of NKp30 have been discovered, and the molecular details of ligand recognition by NKp30 are poorly understood. Within the current study, we found that the ectodomain of NKp30 forms functional homo-oligomers that mediate high affinity binding to its corresponding cellular ligand B7-H6. Notably, this homo-oligomerization is strongly promoted by the stalk domain of NKp30. Based on these data, we suggest that homo-oligomerization of NKp30 in the plasma membrane of NK cells, which might be favored by IL-2-dependent up-regulation of NKp30 expression, provides a way to improve recognition and lysis of target cells by NK cells.

To truly understand bioenergetic processes such as ATP synthesis, membrane-bound substrate transport or flagellar rotation, systems need to be analysed in a cellular context. Cryo-ET (cryo-electron tomography) is an essential part of this process, as it is currently the only technique which can directly determine the spatial organization of proteins at the level of both the cell and the individual protein complexes. The need to assess bioenergetic processes at a cellular level is becoming more and more apparent with the increasing interest in mitochondrial diseases. In recent years, cryo-ET has contributed significantly to our understanding of the molecular organization of mitochondria and chloroplasts. The present mini-review first describes the technique of cryo-ET and then discusses its role in membrane bioenergetics specifically in chloroplasts and mitochondrial research.

F1Fo-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology.

We used electron cryotomography of mitochondrial membranes from wild-type and mutant Saccharomyces cerevisiae to investigate the structure and organization of ATP synthase dimers in situ. Subtomogram averaging of the dimers to 3.7 nm resolution revealed a V-shaped structure of twofold symmetry, with an angle of 86° between monomers. The central and peripheral stalks are well resolved. The monomers interact within the membrane at the base of the peripheral stalks. In wild-type mitochondria ATP synthase dimers are found in rows along the highly curved cristae ridges, and appear to be crucial for membrane morphology. Strains deficient in the dimer-specific subunits e and g or the first transmembrane helix of subunit 4 lack both dimers and lamellar cristae. Instead, cristae are either absent or balloon-shaped, with ATP synthase monomers distributed randomly in the membrane. Computer simulations indicate that isolated dimers induce a plastic deformation in the lipid bilayer, which is partially relieved by their side-by-side association. We propose that the assembly of ATP synthase dimer rows is driven by the reduction in the membrane elastic energy, rather than by direct protein contacts, and that the dimer rows enable the formation of highly curved ridges in mitochondrial cristae.

Membrane proteins account for about 20–30 % of the protein-encoding genes in the genomes of all living organisms.2 Their fundamental importance in human health and disease is underlined by the fact that many drugs in current use act on them.3 Functional and structural studies of membrane proteins are therefore of increasing importance but more difficult and demanding than studies with soluble proteins, because membrane proteins function in the lipid bilayer of cell membranes. For in vitro studies, the proteins are first extracted from the membrane by detergent solubilization and then purified in detergent solution. Sensitive membrane proteins are often unstable in detergent solution, but quite stable once they are reconstituted into a lipid bilayer. Moreover, many membrane proteins require a lipid environment for activity. The reconstitution process, in which detergent is replaced by lipid, must be carefully controlled, as otherwise the proteins tend to denature and aggregate.4

Mitochondria are the powerhouses of eukaryotic cells and the main site of ATP synthesis in cells performing aerobic respiration. Located on the cristae of the inner mitochondrial membrane, the ATP synthase uses the energy stored in a transmembrane gradient of protons to power the synthesis of ATP while the respiratory chain complexes replenish the gradient. Using the technique of electron cryo-tomography and subtomogram averaging, we are characterizing how the structure and spatial distribution of membrane proteins influence mitochondrial function. So far we have discovered: 1) the structure of the ATP synthase varies dramatically between different eukaryotic lineages, 2) that in all species studied so far, the ATP synthase form rows of dimers along the highly curved edges of the cristae membranes, 3) that the structure of the rows varies between protozoan and multicellular organisms, and 4) the purpose of the rows appears to be the maintenance of organized inner membrane invaginations. In parallel, we have shown that the proteins of the respiratory chain are located in the flat membrane regions of cristae and that they form species specific supercomplexes. Alongside the cryo-ET studies, we have determined a 6.2A structure of the mitochondrial ATP synthase of Polytomella sp. using single particle cryo-EM. Our structure revealed four highly tilted membrane-intrinsic helices (∼20° to the membrane plane) adjacent to the rotor ring which we assign to the elusive a-subunit. Based on this structure, we present a model describing how protons move through the ATP synthase.

Hydrogen uptake protein regulator (HupR) is a member of the nitrogen regulatory protein C (NtrC) family of response regulators. These proteins activate transcription by RNA polymerase (RNAP) in response to a change in environment. This change is detected through the phosphorylation of their receiver domain as part of a two-component signalling pathway. HupR is an unusual member of this family as it activates transcription when unphosphorylated, and transcription is inhibited by phosphorylation. Also, HupR activates transcription through the more general sigma(70) transcription initiation factor, which does not require activation by ATPase, in contrast to other NtrC family members that utilise sigma(54). Hence, its mode of action is expected to be different from those of the more conventional NtrC family members. We have determined the structures of the unphosphorylated N-terminal receiver domain of wild-type HupR, the mutant HupR(D55E)(N) (which cannot be phosphorylated and down-regulated), and HupR in the presence of the phosphorylation mimic BeF(3)(-). The structures show a typical response regulator fold organised as a dimer whose interface involves alpha4-beta5-alpha5 elements. The interactions across the interface are slightly different between apo and phospho mimics, and these reflect a rearrangement of key conserved residues around the active site aspartate that have been implicated in domain activation in other receiver domain proteins. We also show that the wild-type HupR receiver domain forms a weak dimer in solution, which is strengthened in the presence of the phosphorylation mimic BeF(3)(-). The results indicate many features similar to those that have been observed in other systems, including NtrC (where phosphorylation is activatory), and indicate that recognition properties, which allow HupR to be active in the absence of phosphorylation, lie in the transmission of phosphorylation signals through the linker region to the other domains of the protein.

Rotary ATPases are energy-converting nanomachines found in the membranes of all living organisms. The mechanism by which proton translocation through the membrane drives ATP synthesis, or how ATP hydrolysis generates a transmembrane proton gradient, has been unresolved for decades because the structure of a critical subunit in the membrane was unknown. Electron cryomicroscopy (cryoEM) studies of two rotary ATPases have now revealed a hairpin of long, horizontal, membrane-intrinsic α-helices in the a-subunit next to the c-ring rotor. The horizontal helices create a pair of aqueous half-channels in the membrane that provide access to the proton-binding sites in the rotor ring. These recent findings help to explain the highly conserved mechanism of ion translocation by rotary ATPases.

SummaryThe mitochondrial contact site and cristae organizing system (MICOS) is a conserved multi-subunit complex crucial for maintaining the characteristic architecture of mitochondria. Studies with deletion mutants identified Mic10 and Mic60 as core subunits of MICOS. Mic60 has been studied in detail; however, topogenesis and function of Mic10 are unknown. We report that targeting of Mic10 to the mitochondrial inner membrane requires a positively charged internal loop, but no cleavable presequence. Both transmembrane segments of Mic10 carry a characteristic four-glycine motif, which has been found in the ring-forming rotor subunit of F1Fo-ATP synthases. Overexpression of Mic10 profoundly alters the architecture of the inner membrane independently of other MICOS components. The four-glycine motifs are dispensable for interaction of Mic10 with other MICOS subunits but are crucial for the formation of large Mic10 oligomers. Our studies identify a unique role of Mic10 oligomers in promoting the formation of inner membrane crista junctions.

We have used a combination of electron cryo-tomography, subtomogram averaging, and electron crystallographic image processing to analyse the structure of intact bovine F1Fo ATP synthase in 2D membrane crystals. ATPase assays and mass spectrometry analysis of the 2D crystals confirmed that the enzyme complex was complete and active. The structure of the matrix-exposed region was determined at 24 Å resolution by subtomogram averaging and repositioned into the tomographic volume to reveal the crystal packing. F1Fo ATP synthase complexes are inclined by 16° relative to the crystal plane, resulting in a zigzag topology of the membrane and indicating that monomeric bovine heart F1Fo ATP synthase by itself is sufficient to deform lipid bilayers. This local membrane curvature is likely to be instrumental in the formation of ATP synthase dimers and dimer rows, and thus for the shaping of mitochondrial cristae. DOI: http://dx.doi.org/10.7554/eLife.06119.001, eLife digest Cells use a molecule called adenosine triphosphate (or ATP for short) to power many processes that are vital for life. Animals, plants, and fungi primarily make their ATP in a specialised compartment called the mitochondrion, which is found inside their cells. The mitochondrion is often referred to as the powerhouse of cells as it captures and stores the energy that animals gain from eating food in the molecule ATP. Other enzymes in the cell break apart ATP to release the stored energy, which they use to power various cellular processes. The interior architecture of the mitochondrion includes a highly folded inner membrane where electrical energy is transformed into chemical energy. The tight folding of the inner membrane is thought to make this process more efficient. An enzyme named ATP synthase performs the final steps of the energy transformation process by producing ATP (ATP synthase literally means ‘ATP maker’). This enzyme sits in pairs along the edges of the inner membrane folds. This raises the question: does the ATP synthase cause the membrane to fold or does this enzyme just ‘prefer’ these folded edges (which are instead caused by something else inside the mitochondrion)? To investigate this question, Jiko, Davies et al. extracted ATP synthase from the mitochondria of cow hearts and mixed them with modified fat molecules to form a ‘2D membrane crystal’: a membrane containing an ordered pattern of enzymes. An electron microscope was used to generate a three-dimensional volume of the 2D membrane crystal via a process similar to a MRI or CAT scan that one might have in hospital. In the three-dimensional volume of the membrane crystal, Jiko, Davies et al. discovered that instead of being flat as expected, the membrane of the 2D membrane crystal was rippled and that this ripple was caused by the membrane-embedded part of the ATP synthase. The geometry of the ripple exactly matched half of the bend at the edge of the membrane folds in the mitochondrion. Therefore, Jiko, Davies et al. concluded that a pair of ATP synthases, as found in mitochondria, was responsible for defining the tight folds of the inner mitochondrial membrane. DOI: http://dx.doi.org/10.7554/eLife.06119.002

Bacterial RNA polymerases (RNAPs) are capable of producing full-length transcripts in the absence of additional factors using in vitro assays. However, in vivo RNAP can become stalled during the elongation phase of transcription due to the presence of various sequence motifs. Subsequently, a host of elongation factors are required to modulate the activity of RNAP. NusA, the most intensively studied elongation factor, plays a role in increasing RNAP pausing and termination. Conversely, it is also important in transcription of rRNA where it functions as an anti-termination factor, helping to ensure only full-length transcripts are produced. Here we show that NusA is closely associated with RNAP within the bacterial nucleoid and that it is preferentially recruited to sites of rRNA synthesis. In vivo and in vitro analyses indicate this results in a change in stoichiometry of NusA:RNAP from 1:1 to approximately 2:1 at the subcellular sites of rRNA synthesis. A model is presented showing how the ratio of NusA:RNAP could affect the activity of the elongation complex so that it functions as an anti-terminator complex during rRNA synthesis.

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F 1 heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.

HupR is a response regulator that controls the synthesis of the membrane-bound [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus. The protein belongs to the NtrC subfamily of response regulators and is the second protein of a two-component system. We have crystallized the full-length protein HupR in the unphosphorylated state in two dimensions using the lipid monolayer technique. The 3D structure of negatively stained HupR was calculated to a resolution of approximately 23 A from tilted electron microscope images. HupR crystallizes as a dimer, and forms an elongated V-shaped structure with extended arms. The dimensions of the dimer are about 80 A length, 40 A width and 85 A thick. The HupR monomer consists of three domains, N-terminal receiver domain, central domain and C-terminal DNA-binding domain. We have fitted the known 3D structure of the central domain from NtrC1 Aquifex aeolicus protein into our 3D model; we propose that contact between the dimers is through the central domain. The N-terminal domain is in contact with the lipid monolayer and is situated on the top of the V-shaped structure. The central domain alone has been expressed and purified; it forms a pentamer in solution and lacks ATPase activity.

Bacterial RNA polymerases (RNAPs) are capable of producing full-length transcripts in the absence of additional factors using in vitro assays. However, in vivo RNAP can become stalled during the elongation phase of transcription due to the presence of various sequence motifs. Subsequently, a host of elongation factors are required to modulate the activity of RNAP. NusA, the most intensively studied elongation factor, plays a role in increasing RNAP pausing and termination. Conversely, it is also important in transcription of rRNA where it functions as an anti-termination factor, helping to ensure only full-length transcripts are produced. Here we show that NusA is closely associated with RNAP within the bacterial nucleoid and that it is preferentially recruited to sites of rRNA synthesis. In vivo and in vitro analyses indicate this results in a change in stoichiometry of NusA:RNAP from 1:1 to approximately 2:1 at the subcellular sites of rRNA synthesis. A model is presented showing how the ratio of NusA:RNAP could affect the activity of the elongation complex so that it functions as an anti-terminator complex during rRNA synthesis.

InBacillus subtilis, RNA polymerase becomes concentrated into regions of the nucleoid called transcription foci. With green fluorescent protein-tagged RNA polymerase, these structures are only observed at higher growth rates and have been shown to represent the sites of rRNA synthesis. There are 10 rRNA (rrn) operons distributed around nearly half of the chromosome. In this study we analyzed therrncomposition of transcription foci with fluorescently tagged loci and showed that they comprise the origin-proximal operonrrnObut not the more dispersedrrnEorrrnD. This suggests that transcription foci comprise only the seven origin-proximal operonsrrnO,rrnA,rrnJ,rrnW,rrnI,rrnH, andrrnG. These results have important implications for our understanding of microbial chromosome structure.

Mitochondria Cristae Karen M. Davies, Bertram Daum, Claudio Anselmi, Jose D. Faraldo-Gomez, Werner Kuhlbrandt Max Planck Institute of Biophysics, Department of Structural Biology, Max-von-Laue Str. 3, 60438 Frankfurt am Main, Germany. Max Planck Institute of Biophysics, Department of Theoretical Molecular Biophysics Group and Cluster of Excellence ‘Macromolecular Complexes’, Max-von-Laue Str. 3, 60438 Frankfurt am Main, Germany.