Molecular basis of ß-arrestin coupling to formoterol-bound ß1-adrenoceptor.

The ß1-adrenoceptor (ß1AR) is a G-protein-coupled receptor (GPCR) that couples1 to the heterotrimeric G protein Gs. G-protein-mediated signalling is terminated by phosphorylation of the C terminus of the receptor by GPCR kinases (GRKs) and by coupling of ß-arrestin 1 (ßarr1, also known as arrestin 2), which displaces Gs and induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins-known as biased agonism3-is important in drug development, because the therapeutic effect may arise from only one signalling cascade, whereas the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, here we determined the cryo-electron microscopy structure of the ß1AR-ßarr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound ß1AR coupled to the G-protein-mimetic nanobody6 Nb80. ßarr1 couples to ß1AR in a manner distinct to that7 of Gs coupling to ß2AR-the finger loop of ßarr1 occupies a narrower cleft on the intracellular surface, and is closer to transmembrane helix H7 of the receptor when compared with the C-terminal α5 helix of Gs. The conformation of the finger loop in ßarr1 is different from that adopted by the finger loop of visual arrestin when it couples to rhodopsin8. ß1AR coupled to ßarr1 shows considerable differences in structure compared with ß1AR coupled to Nb80, including an inward movement of extracellular loop 3 and the cytoplasmic ends of H5 and H6. We observe weakened interactions between formoterol and two serine residues in H5 at the orthosteric binding site of ß1AR, and find that formoterol has a lower affinity for the ß1AR-ßarr1 complex than for the ß1AR-Gs complex. The structural differences between these complexes of ß1AR provide a foundation for the design of small molecules that could bias signalling in the ß-adrenoceptors.

The ontogeny of disparity in Cupressaceae seed cones.

Ontogenetic shape change has long been recognized to be important in generating patterns of morphological diversity and may be especially important in plant reproductive structures. We explore how seed cone disparity in Cupressaceae changes over ontogeny by comparing pollination-stage and mature cones. We sampled cones at pollen and seed release and measured cone scales using basic morphometric shape variables. We used multivariate statistical methods, particularly hypervolume overlap calculations, to measure morphospace occupation and disparity. Cone scales at both pollination and maturity exhibit substantial variability, although the disparity is greater at maturity. Mature cone scales are also more clustered in trait space, showing less overlap with other taxa than at pollination. These patterns reflect two growth strategies that generate closed cones over maturation, either through thin laminar scales or relatively thick, peltate scales, resulting in two distinct regions of morphospace occupation. Disparity patterns in Cupressaceae seed cones change over ontogeny, reflecting shifting functional demands that require specific patterns of cone scale growth. The evolution of Cupressaceae reproductive disparity therefore represents selection for trajectories of ontogenetic shape change, a phenomenon that should be widespread across seed plants.

Hidden functional complexity in the flora of an early land ecosystem.

The first land ecosystems were composed of organisms considered simple in nature, yet the morphological diversity of their flora was extraordinary. The biological significance of this diversity remains a mystery largely due to the absence of feasible study approaches. To study the functional biology of Early Devonian flora, we have reconstructed extinct plants from fossilised remains in silico. We explored the morphological diversity of sporangia in relation to their mechanical properties using finite element method. Our approach highlights the impact of sporangia morphology on spore dispersal and adaptation. We discovered previously unidentified innovations among early land plants, discussing how different species might have opted for different spore dispersal strategies. We present examples of convergent evolution for turgor pressure resistance, achieved by homogenisation of stress in spherical sporangia and by torquing force in Tortilicaulis-like specimens. In addition, we show a potential mechanism for stress-assisted sporangium rupture. Our study reveals the deceptive complexity of this seemingly simple group of organisms. We leveraged the quantitative nature of our approach and constructed a fitness landscape to understand the different ecological niches present in the Early Devonian Welsh Borderland flora. By connecting morphology to functional biology, these findings facilitate a deeper understanding of the diversity of early land plants and their place within their ecosystem.

Quantifying the complexity of plant reproductive structures reveals a history of morphological and functional integration.

Vascular plant reproductive structures have undoubtedly become more complex through time, evolving highly differentiated parts that interact in specialized ways. But quantifying these patterns at broad scales is challenging because lineages produce disparate reproductive structures that are often difficult to compare and homologize. We develop a novel approach for analysing interactions within reproductive structures using networks, treating component parts as nodes and a suite of physical and functional interactions among parts as edges. We apply this approach to the plant fossil record, showing that interactions have generally increased through time and that the concentration of these interactions has shifted towards differentiated surrounding organs, resulting in more compact, functionally integrated structures. These processes are widespread across plant lineages, but their extent and timing vary with reproductive biology; in particular, seed-producing structures show them more strongly than spore or pollen-producing structures. Our results demonstrate that major reproductive innovations like the origin of seeds and angiospermy were associated with increased integration through greater interactions among parts. But they also reveal that for certain groups, particularly Mesozoic gymnosperms, millions of years elapsed between the origin of reproductive innovations and increased interactions among parts within their reproductive structures.

Critical success factors for school-based integrated health care models: Learnings from an Australian example.

ISSUES ADDRESSED: Integrated school-based health services have the potential to address the unmet health needs of children experiencing disadvantage, yet these models remain poorly evaluated. The current article examines an integrated social and health care hub located on the grounds of a regional Australian public primary school, the Our Mia Mia Wellbeing Hub, to identify critical success factors for this service and others like it. METHODS: Semi-structured qualitative interviews were conducted with N = 55 multi-sector stakeholders comprising parents, students, school staff, social and health care providers, and local Aboriginal community members. Interview transcripts were analysed according to a grounded theory approach. RESULTS: Six themes emerged from the analysis, reflecting important success factors for the model: service accessibility; service coordination; integration of education and health systems; trust; community partnerships; and perceptions of health. CONCLUSIONS: Findings highlighted Our Mia Mia as a promising model of care, yet also revealed important challenges for the service as it responds to the varied priorities of the stakeholders it serves. SO WHAT?: Through capturing the perspectives of a large number of stakeholders, the current study provides valuable insight into key challenges and success factors for Our Mia Mia; these learnings can guide the development of other emerging school-based health services and integrated care hubs.

Structure of the adenosine A(2A) receptor bound to an engineered G protein.

G-protein-coupled receptors (GPCRs) are essential components of the signalling network throughout the body. To understand the molecular mechanism of G-protein-mediated signalling, solved structures of receptors in inactive conformations and in the active conformation coupled to a G protein are necessary. Here we present the structure of the adenosine A(2A) receptor (A(2A)R) bound to an engineered G protein, mini-Gs, at 3.4 Å resolution. Mini-Gs binds to A(2A)R through an extensive interface (1,048 Å2) that is similar, but not identical, to the interface between Gs and the ß2-adrenergic receptor. The transition of the receptor from an agonist-bound active-intermediate state to an active G-protein-bound state is characterized by a 14 Å shift of the cytoplasmic end of transmembrane helix 6 (H6) away from the receptor core, slight changes in the positions of the cytoplasmic ends of H5 and H7 and rotamer changes of the amino acid side chains Arg3.50, Tyr5.58 and Tyr7.53. There are no substantial differences in the extracellular half of the receptor around the ligand binding pocket. The A(2A)R-mini-Gs structure highlights both the diversity and similarity in G-protein coupling to GPCRs and hints at the potential complexity of the molecular basis for G-protein specificity.

The structure of the catalytic domain of the ATP synthase from Mycobacterium smegmatis is a target for developing antitubercular drugs.

The crystal structure of the F1-catalytic domain of the adenosine triphosphate (ATP) synthase has been determined from Mycobacterium smegmatis which hydrolyzes ATP very poorly. The structure of the α3ß3-component of the catalytic domain is similar to those in active F1-ATPases in Escherichia coli and Geobacillus stearothermophilus However, its ε-subunit differs from those in these two active bacterial F1-ATPases as an ATP molecule is not bound to the two α-helices forming its C-terminal domain, probably because they are shorter than those in active enzymes and they lack an amino acid that contributes to the ATP binding site in active enzymes. In E. coli and G. stearothermophilus, the α-helices adopt an "up" state where the α-helices enter the α3ß3-domain and prevent the rotor from turning. The mycobacterial F1-ATPase is most similar to the F1-ATPase from Caldalkalibacillus thermarum, which also hydrolyzes ATP poorly. The ßE-subunits in both enzymes are in the usual "open" conformation but appear to be occupied uniquely by the combination of an adenosine 5'-diphosphate molecule with no magnesium ion plus phosphate. This occupation is consistent with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic products are probably the basis of the inhibition of ATP hydrolysis. It can be envisaged that specific as yet unidentified small molecules might bind to the F1 domain in Mycobacterium tuberculosis, prevent ATP synthesis, and inhibit the growth of the pathogen.

Early Cretaceous abietoid Pinaceae from Mongolia and the history of seed scale shedding.

PREMISE: Seed cones of extant Pinaceae exhibit two mechanisms of seed release. In "flexers" the cone scales remain attached to the central axis, while flexing and separating from each other to release the seeds. In "shedders" scales are shed from the axis, with the seeds either remaining attached to the scale or becoming detached. The early fossil history of Pinaceae from the Jurassic to Early Cretaceous is dominated by flexing seed cones, while the systematic information provided by shedding fossil cones has been overlooked and rarely integrated with data based on compression and permineralized specimens. We describe the earliest and best-documented evidence of a "shedder" seed cone from the Aptian-Albian of Mongolia. METHODS: Lignite samples from Tevshiin Govi locality were disaggregated in water, washed, and dried in air. Fossils were compared to material of extant Pinaceae using LM and CT scans. RESULTS: Lepidocasus mellonae gen. et sp. nov. is characterized by a seed cone that disarticulated at maturity and shed obovate bract-scale complexes that have a distinctive ribbed surface and an abaxial surface covered with abundant trichomes. The ovuliferous scale has ca. 30-40 resin canals, but only scarce xylem near the attachment to the cone axis. Resin vesicles are present in the seed integument. Phylogenetic analysis places Lepidocasus as sister to extant Cedrus within the abietoid grade. CONCLUSIONS: The exquisite preservation of the trichomes in L. mellonae raises questions about their potential ecological function in the cones of fossil and living Pinaceae. Lepidocasus mellonae also shows that a shedding dispersal syndrome, a feature that has often been overlooked, evolved early in the history of Pinaceae during the Early Cretaceous.

ATP synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites.

The structures and functions of the components of ATP synthases, especially those subunits involved directly in the catalytic formation of ATP, are widely conserved in metazoans, fungi, eubacteria, and plant chloroplasts. On the basis of a map at 32.5-Å resolution determined in situ in the mitochondria of Trypanosoma brucei by electron cryotomography, it has been proposed that the ATP synthase in this species has a noncanonical structure and different catalytic sites in which the catalytically essential arginine finger is provided not by the α-subunit adjacent to the catalytic nucleotide-binding site as in all species investigated to date, but rather by a protein, p18, found only in the euglenozoa. A crystal structure at 3.2-Å resolution of the catalytic domain of the same enzyme demonstrates that this proposal is incorrect. In many respects, the structure is similar to the structures of F1-ATPases determined previously. The α3ß3-spherical portion of the catalytic domain in which the three catalytic sites are found, plus the central stalk, are highly conserved, and the arginine finger is provided conventionally by the α-subunits adjacent to each of the three catalytic sites found in the ß-subunits. Thus, the enzyme has a conventional catalytic mechanism. The structure differs from previous described structures by the presence of a p18 subunit, identified only in the euglenozoa, associated with the external surface of each of the three α-subunits, thereby elaborating the F1-domain. Subunit p18 is a pentatricopeptide repeat (PPR) protein with three PPRs and appears to have no function in the catalytic mechanism of the enzyme.

Accumulation over evolutionary time as a major cause of biodiversity hotspots in conifers.

Biodiversity hotspots are important for understanding how areas of high species richness form, but disentangling the processes that produce them is difficult. We combine geographical ranges, phylogenetic relationships and trait data for 606 conifer species in order to explore the mechanisms underlying richness hotspot formation. We identify eight richness hotspots that overlap known centres of plant endemism and diversity, and find that conifer richness hotspots occur in mountainous areas within broader regions of long-term climate stability. Conifer hotspots are not unique in their species composition, traits or phylogenetic structure; however, a large percentage of their species are not restricted to hotspots and they rarely show either a preponderance of new radiating lineages or old relictual lineages. We suggest that conifer hotspots have primarily formed as a result of lineages accumulating over evolutionary time scales in stable mountainous areas rather than through high origination, preferential retention of relictual lineages or radiation of species with unique traits, although such processes may contribute to nuanced differences among hotspots. Conifers suggest that a simple accumulation of regional diversity can generate high species richness without additional processes and that geography rather than biology may play a primary role in hotspot formation.

Not all 'pine cones' flex: functional trade-offs and the evolution of seed release mechanisms.

Seed dispersal is critical for plants, but the evolution of mechanisms that actually release seeds from their parents is not well understood. We use the reproductive cones of conifers, specifically the Pinaceae clade, to explore the factors driving the evolution of different release mechanisms in plants. We combine comparative anatomical and phylogenetic analyses to test whether fundamental trade-offs in the mechanical and hydraulic properties of vasculature underlie the evolution of two seed release mechanisms: cone scale flexion and cone scale shedding. We then test whether these mechanisms are linked with differences in seed size, dispersal syndrome and reproductive allocation. Cone scale xylem in flexing species is tough, but poorly conductive. Xylem in shedding species is less extensive, fragile and highly conductive; its thin-walled tracheids allow scales to easily fracture at maturity. Shedding is also consistently associated with large, densely packed seeds. Pinaceae cones exploit a well-known trade-off in xylem mechanical strength vs hydraulic efficiency to generate release mechanisms that allow seeds of various sizes to leave the protecting cone. The linkage among release mechanisms, vascular anatomy and seed traits illustrates how a wide variety of selective pressures may influence the function and physiology of reproductive structures.

Functional diversity and convergence in the evolution of plant reproductive structures.

Background and Aims: Structures that simultaneously perform many functional roles are likely to show a variety of morphological solutions to these demands, and thus probably exhibit high morphological disparity. In contrast, specialization for a few simple functions should result in a more limited suite of morphologies. We explore this idea using lycopsid reproductive structures, which, throughout their history, have performed a limited set of functional roles compared with the reproductive structures of other plant groups such as seed plants. Methods: We scored living and fossil lycopsid taxa for 18 discrete character measurements and several continuous traits, including sporangium size, supporting axis diameter, and strobilus length and width. We used the discrete characters to construct a multivariate morphospace for lycopsid reproductive morphology through time, and the continuous characters to test whether fossil and extant lycopsids show similar patterns of tissue allocation within reproductive structures. Results: Lycopsids occupy similar areas of reproductive morphospace and show similar patterns of tissue allocation over most of their history, alternating between diffuse fertile zones with leaf-like sporophylls and compact strobili with specialized sporophylls that allow sporangia to be closely packed while also protected during their development. Growth habit also plays an important role in lycopsid reproductive evolution, broadly influencing the size and shape of reproductive structures. Conclusions: Lycopsid reproductive structures are primarily specialized for densely packaging sporangia, and are consistent with the idea that performing limited functional roles is associated with reduced morphological disparity. Morphologies similar to lycopsid strobili are also found in other groups with simple, wind-dispersed propagules, suggesting that the same processes occur across plant lineages.

Neurocritical care of high-risk infants during inter-hospital transport.

The centralisation of neonatal intensive care in recent years has improved mortality, particularly of extremely preterm infants, but similar improvements in morbidity, such as neurodevelopmental impairment, have not been seen. Integral to the success of centralisation are specialised neonatal transport teams who provide intensive care prior to and during retrieval of high-risk neonates when in-utero transfer has not been possible. Neonatal retrieval aims to stabilise the clinical condition and then transfer the neonate during a high-risk period for patient. Transport introduces the hazards of noise and vibration; acceleration and deceleration forces; additional handling and temperature fluctuations. The transport team must stabilise the infant fully prior to transport as when on the move they are limited by space and movement to effectively attend to clinical deterioration. Inborn infants have better neurodevelopmental outcome compared with the outborn and aetiology of this seems to be multifactorial with the impact of transport itself during critical illness, remaining unclear. To improve the neurological outcomes for transported infants, it seems imperative to integrate the advancing intensive care neuromonitoring tools into the transport milieu. This review examines current inter-hospital transport neuromonitoring and how new modalities might be applied to the neurocritical care delivered by specialist transport teams.

Servo-controlled cooling during neonatal transport for babies with hypoxic-ischaemic encephalopathy is practical and beneficial: Experience from a large UK neonatal transport service.

AIM: Servo-controlled therapeutic hypothermia is a routine therapy for babies with hypoxic-ischaemic encephalopathy in the neonatal unit and is delivered in designated cooling centres. It is increasingly being used during neonatal transport in the UK to deliver this therapy in a timelier manner for babies not born in a cooling centre. Prior to the implementation of this treatment, passive cooling alone was used in transport. Comparison of passive and servo-controlled cooling during neonatal transfers with reference to: (i) the proportion of babies in the therapeutic range (33-34°C) at three time points during the transport mission (arrival of the team at the referring unit, departure of the team from the referring unit and at the completion of transport); (ii) the proportion of babies overcooled at any point once the transport team was present (<33°C); and (iii) duration of phases of the transfer to evaluate the impact of active cooling on how long it takes to undertake transfer. METHODS: This was a retrospective observational study comparing babies with passive and servo-controlled hypothermia (January 2015 to May 2016) following introduction of the servo-controlled cooling mattress. RESULTS: A total of 48 patients were treated with hypothermia in transit (29 passive, 19 servo-controlled). The median gestational age (GA) was 40 weeks (interquartile range: 39-41) and mean birthweight (BW) 3420 g (standard deviation 600 g); there was no differences in GA, BW and clinical characteristics between the groups. There was a statistically significant difference in the temperature at the end of the transport, where 94% (n = 18) of babies who received servo-controlled cooling were in the target temperature in comparison with only 65% (n = 19) of the passive cooling group babies (P = 0.045). In addition, none of the babies in the servo-controlled group were warm (>34°C) at the end of the transfer. Babies who underwent servo-controlled cooling are more likely to maintain a target temperature (33-34°C) (17 (89%) vs. 17 (58%), P = 0.021); in particular, there is less overcooling (<33°C) in this group (2 (10%) vs. 15 (51%), P = 0.004). Total mission time was not significantly different. CONCLUSION: The use of servo-controlled cooling devices during neonatal transport improves the ability to maintain the baby's temperature within the target range (33-34°C) with less overcooling.

Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum.

The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a "down" state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an "up" state, where the α-helices, devoid of ATP, enter the α3ß3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme's hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3ß3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the ßE-catalytic site is in the usual "open" conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis.

Why are the seed cones of conifers so diverse at pollination?

Background and Aims: Form and function relationships in plant reproductive structures have long fascinated biologists. Although the intricate associations between specific pollinators and reproductive morphology have been widely explored among animal-pollinated plants, the evolutionary processes underlying the diverse morphologies of wind-pollinated plants remain less well understood. Here we study how this diversity may have arisen by focusing on two conifer species in the pine family that have divergent reproductive cone morphologies at pollination. Methods: Standard histology methods, artificial wind pollination assays and phylogenetic analyses were used in this study. Key Results: A detailed study of cone ontogeny in these species reveals that variation in the rate at which their cone scales mature means that pollination occurs at different stages in their development, and thus in association with different specific morphologies. Pollination experiments nevertheless indicate that both species effectively capture pollen. Conclusions: In wind-pollinated plants, morphological diversity may result from simple variation in development among lineages rather than selective pressures for any major differences in function or performance. This work also illustrates the broader importance of developmental context in understanding plant form and function relationships; because plant reproductive structures perform many different functions over their lifetime, subtle differences in development may dramatically alter the specific morphologies that they use to meet these demands.

An overview of extant conifer evolution from the perspective of the fossil record.

PREMISE OF THE STUDY: Conifers are an important living seed plant lineage with an extensive fossil record spanning more than 300 million years. The group therefore provides an excellent opportunity to explore congruence and conflict between dated molecular phylogenies and the fossil record. METHODS: We surveyed the current state of knowledge in conifer phylogenetics to present a new time-calibrated molecular tree that samples ~90% of extant species diversity. We compared phylogenetic relationships and estimated divergence ages in this new phylogeny with the paleobotanical record, focusing on clades that are species-rich and well known from fossils. KEY RESULTS: Molecular topologies and estimated divergence ages largely agree with the fossil record in Cupressaceae, conflict with it in Araucariaceae, and are ambiguous in Pinaceae and Podocarpaceae. Molecular phylogenies provide insights into some fundamental questions in conifer evolution, such as the origin of their seed cones, but using them to reconstruct the evolutionary history of specific traits can be challenging. CONCLUSIONS: Molecular phylogenies are useful for answering deep questions in conifer evolution if they depend on understanding relationships among extant lineages. Because of extinction, however, molecular datasets poorly sample diversity from periods much earlier than the Late Cretaceous. This fundamentally limits their utility for understanding deep patterns of character evolution and resolving the overall pattern of conifer phylogeny.

Structure of ATP synthase from Paracoccus denitrificans determined by X-ray crystallography at 4.0 Å resolution.

The structure of the intact ATP synthase from the α-proteobacterium Paracoccus denitrificans, inhibited by its natural regulatory ζ-protein, has been solved by X-ray crystallography at 4.0 Å resolution. The ζ-protein is bound via its N-terminal α-helix in a catalytic interface in the F1 domain. The bacterial F1 domain is attached to the membrane domain by peripheral and central stalks. The δ-subunit component of the peripheral stalk binds to the N-terminal regions of two α-subunits. The stalk extends via two parallel long α-helices, one in each of the related b and b' subunits, down a noncatalytic interface of the F1 domain and interacts in an unspecified way with the a-subunit in the membrane domain. The a-subunit lies close to a ring of 12 c-subunits attached to the central stalk in the F1 domain, and, together, the central stalk and c-ring form the enzyme's rotor. Rotation is driven by the transmembrane proton-motive force, by a mechanism where protons pass through the interface between the a-subunit and c-ring via two half-channels in the a-subunit. These half-channels are probably located in a bundle of four α-helices in the a-subunit that are tilted at ∼30° to the plane of the membrane. Conserved polar residues in the two α-helices closest to the c-ring probably line the proton inlet path to an essential carboxyl group in the c-subunit in the proton uptake site and a proton exit path from the proton release site. The structure has provided deep insights into the workings of this extraordinary molecular machine.

How release of phosphate from mammalian F1-ATPase generates a rotary substep.

The rotation of the central stalk of F1-ATPase is driven by energy derived from the sequential binding of an ATP molecule to its three catalytic sites and the release of the products of hydrolysis. In human F1-ATPase, each 360° rotation consists of three 120° steps composed of substeps of about 65°, 25°, and 30°, with intervening ATP binding, phosphate release, and catalytic dwells, respectively. The F1-ATPase inhibitor protein, IF1, halts the rotary cycle at the catalytic dwell. The human and bovine enzymes are essentially identical, and the structure of bovine F1-ATPase inhibited by IF1 represents the catalytic dwell state. Another structure, described here, of bovine F1-ATPase inhibited by an ATP analog and the phosphate analog, thiophosphate, represents the phosphate binding dwell. Thiophosphate is bound to a site in the α(E)ß(E)-catalytic interface, whereas in F1-ATPase inhibited with IF1, the equivalent site is changed subtly and the enzyme is incapable of binding thiophosphate. These two structures provide a molecular mechanism of how phosphate release generates a rotary substep as follows. In the active enzyme, phosphate release from the ß(E)-subunit is accompanied by a rearrangement of the structure of its binding site that prevents released phosphate from rebinding. The associated extrusion of a loop in the ß(E)-subunit disrupts interactions in the α(E)ß(E-)catalytic interface and opens it to its fullest extent. Other rearrangements disrupt interactions between the γ-subunit and the C-terminal domain of the α(E)-subunit. To restore most of these interactions, and to make compensatory new ones, the γ-subunit rotates through 25°-30°.