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While depolarization of the neuronal membrane is known to evoke the neurotransmitter release from synaptic vesicles, hyperpolarization is regarded as a resting state of chemical neurotransmission. Here, we report that hyperpolarizing neurons can actively signal neural information by employing undocked hemichannels. We show that UNC-7, a member of the innexin family in Caenorhabditis elegans, functions as a hemichannel in thermosensory neurons and transmits temperature information from the thermosensory neurons to their postsynaptic interneurons. By monitoring neural activities in freely behaving animals, we find that hyperpolarizing thermosensory neurons inhibit the activity of the interneurons and that UNC-7 hemichannels regulate this process. UNC-7 is required to control thermotaxis behavior and functions independently of synaptic vesicle exocytosis. Our findings suggest that innexin hemichannels mediate neurotransmission from hyperpolarizing neurons in a manner that is distinct from the synaptic transmission, expanding the way of neural circuitry operations.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Neuronas , Transmisión Sináptica , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Conexinas/metabolismo , Conexinas/genética , Interneuronas/metabolismo , Proteínas de la Membrana , Neuronas/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Taxia/fisiologíaRESUMEN
Hydroxycarboxylic acid receptors (HCA) are expressed in various tissues and immune cells. HCA2 and its agonist are thus important targets for treating inflammatory and metabolic disorders. Only limited information is available, however, on the active-state binding of HCAs with agonists. Here, we present cryo-EM structures of human HCA2-Gi and HCA3-Gi signaling complexes binding with multiple compounds bound. Agonists were revealed to form a salt bridge with arginine, which is conserved in the HCA family, to activate these receptors. Extracellular regions of the receptors form a lid-like structure that covers the ligand-binding pocket. Although transmembrane (TM) 6 in HCAs undergoes dynamic conformational changes, ligands do not directly interact with amino acids in TM6, suggesting that indirect signaling induces a slight shift in TM6 to activate Gi proteins. Structural analyses of agonist-bound HCA2 and HCA3 together with mutagenesis and molecular dynamics simulation provide molecular insights into HCA ligand recognition and activation mechanisms.
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Aminoácidos , Transducción de Señal , Humanos , Ligandos , Aminas , ArgininaRESUMEN
Divalent cation block is observed in various tetrameric ion channels. For blocking, a divalent cation is thought to bind in the ion pathway of the channel, but such block has not yet been directly observed. So, the behaviour of these blocking divalent cations remains still uncertain. Here, we elucidated the mechanism of the divalent cation block by reproducing the blocking effect into NavAb, a well-studied tetrameric sodium channel. Our crystal structures of NavAb mutants show that the mutations increasing the hydrophilicity of the inner vestibule of the pore domain enable a divalent cation to stack on the ion pathway. Furthermore, non-equilibrium molecular dynamics simulation showed that the stacking calcium ion repel sodium ion at the bottom of the selectivity filter. These results suggest the primary process of the divalent cation block mechanism in tetrameric cation channels.
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Canales Iónicos , Canales de Sodio , Cationes Bivalentes/metabolismo , Canales de Sodio/metabolismo , Cationes/metabolismo , Mutación , Calcio/metabolismoRESUMEN
Mirogabalin is a novel gabapentinoid drug with a hydrophobic bicyclo substituent on the γ-aminobutyric acid moiety that targets the voltage-gated calcium channel subunit α2δ1. Here, to reveal the mirogabalin recognition mechanisms of α2δ1, we present structures of recombinant human α2δ1 with and without mirogabalin analyzed by cryo-electron microscopy. These structures show the binding of mirogabalin to the previously reported gabapentinoid binding site, which is the extracellular dCache_1 domain containing a conserved amino acid binding motif. A slight conformational change occurs around the residues positioned close to the hydrophobic group of mirogabalin. Mutagenesis binding assays identified that residues in the hydrophobic interaction region, in addition to several amino acid binding motif residues around the amino and carboxyl groups of mirogabalin, are critical for mirogabalin binding. The A215L mutation introduced to decrease the hydrophobic pocket volume predictably suppressed mirogabalin binding and promoted the binding of another ligand, L-Leu, with a smaller hydrophobic substituent than mirogabalin. Alterations of residues in the hydrophobic interaction region of α2δ1 to those of the α2δ2, α2δ3, and α2δ4 isoforms, of which α2δ3 and α2δ4 are gabapentin-insensitive, suppressed the binding of mirogabalin. These results support the importance of hydrophobic interactions in α2δ1 ligand recognition.
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Canales de Calcio , Gabapentina , Humanos , Canales de Calcio/metabolismo , Microscopía por Crioelectrón , Gabapentina/química , Gabapentina/farmacología , LigandosRESUMEN
Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na+ to H+ selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via P-type ATPases using functional and structural analyses to demonstrate that four simultaneous residue substitutions transform the non-gastric H+/K+ pump, a strict H+-dependent electroneutral P-type ATPase, into a bona fide Na+-dependent electrogenic Na+/K+ pump. Conversion of a H+-dependent primary-active transporter into a Na+-dependent one provides a prototype for similar studies of ion-transport proteins. Moreover, we solve the structures of the wild-type non-gastric H+/K+ pump, a suitable drug target to treat cystic fibrosis, and of its Na+/K+ pump-mimicking mutant in two major conformations, providing insight on how Na+ binding drives a concerted mechanism leading to Na+/K+ pump phosphorylation.
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Fibrosis Quística , ATPasas Tipo P , Humanos , Transporte Iónico , Iones , Mutación MissenseRESUMEN
MrgD, a member of the Mas-related G protein-coupled receptor (MRGPR) family, has high basal activity for Gi activation. It recognizes endogenous ligands, such as ß-alanine, and is involved in pain and itch signaling. The lack of a high-resolution structure for MrgD hinders our understanding of whether its activation is ligand-dependent or constitutive. Here, we report two cryo-EM structures of the MrgD-Gi complex in the ß-alanine-bound and apo states at 3.1 Å and 2.8 Å resolution, respectively. These structures show that ß-alanine is bound to a shallow pocket at the extracellular domains. The extracellular half of the sixth transmembrane helix undergoes a significant movement and is tightly packed into the third transmembrane helix through hydrophobic residues, creating the active form. Our structures demonstrate a structural basis for the characteristic ligand recognition of MrgD. These findings provide a framework to guide drug designs targeting the MrgD receptor.
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Receptores Acoplados a Proteínas G , Transducción de Señal , Microscopía por Crioelectrón , Ligandos , Receptores Acoplados a Proteínas G/metabolismo , beta-AlaninaRESUMEN
Human stomatin (hSTOM) is a component of the membrane skeleton of erythrocytes that maintains the membrane's shape and stiffness through interconnecting spectrin and actin. hSTOM is a member of the protein family that possesses a single stomatin/prohibitin/flotillin/HflK (SPFH) domain at the center of the molecule. Although SPFH domain proteins are widely distributed from archaea to mammals, the detailed function of the domain remains unclear. In this study, we first determined the solution structure of the SPFH domain of hSTOM (hSTOM(SPFH)) via NMR. The solution structure of hSTOM(SPFH) is essentially identical to the already reported crystal structure of the STOM SPFH domain (mSTOM(SPFH)) of mice, except for the existence of a small hydrophilic pocket on the surface. We identified this pocket as a phosphate-binding site by comparing its NMR spectra with and without phosphate ions. Meanwhile, during the conventional process of protein NMR analysis, we eventually discovered that hSTOM(SPFH) formed a unique solid material after lyophilization. This lyophilized hSTOM(SPFH) sample was moderately slowly dissolved in a physiological buffer. Interestingly, it was resistant to dissolution against the phosphate buffer. We then found that the lyophilized hSTOM(SPFH) formed a fibril-like assembly under electron microscopy. Finally, we succeeded in reproducing this fibril-like assembly of hSTOM(SPFH) using a centrifugal ultrafiltration device, thus demonstrating that the increased protein concentration may promote self-assembly of hSTOM(SPFH) into fibril forms. Our observations may help understand the molecular function of the SPFH domain and its involvement in protein oligomerization as a component of the membrane skeleton. (245 words).
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As specific inhibitors of the gastric proton pump, responsible for gastric acidification, K+-competitive acid blockers (P-CABs) have recently been utilized in the clinical treatment of gastric acid-related diseases in Asia. However, as these compounds have been developed based on phenotypic screening, their detailed binding poses are unknown. We show crystal and cryo-EM structures of the gastric proton pump in complex with four different P-CABs, tegoprazan, soraprazan, PF-03716556 and revaprazan, at resolutions reaching 2.8 Å. The structures describe molecular details of their interactions and are supported by functional analyses of mutations and molecular dynamics simulations. We reveal that revaprazan has a novel binding mode in which its tetrahydroisoquinoline moiety binds deep in the cation transport conduit. The mechanism of action of these P-CABs can now be evaluated at the molecular level, which will facilitate the rational development and improvement of currently available P-CABs to provide better treatment of acid-related gastrointestinal diseases.
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Inhibidores de la Bomba de Protones , Bombas de Protones , Ácido Gástrico/metabolismo , Potasio/metabolismo , Inhibidores de la Bomba de Protones/metabolismo , Inhibidores de la Bomba de Protones/farmacología , Bombas de Protones/metabolismo , EstómagoRESUMEN
Tubulins are critical for the internal organization of eukaryotic cells, and understanding their emergence is an important question in eukaryogenesis. Asgard archaea are the closest known prokaryotic relatives to eukaryotes. Here, we elucidated the apo and nucleotide-bound x-ray structures of an Asgard tubulin from hydrothermal living Odinarchaeota (OdinTubulin). The guanosine 5'-triphosphate (GTP)-bound structure resembles a microtubule protofilament, with GTP bound between subunits, coordinating the "+" end subunit through a network of water molecules and unexpectedly by two cations. A water molecule is located suitable for GTP hydrolysis. Time course crystallography and electron microscopy revealed conformational changes on GTP hydrolysis. OdinTubulin forms tubules at high temperatures, with short curved protofilaments coiling around the tubule circumference, more similar to FtsZ, rather than running parallel to its length, as in microtubules. Thus, OdinTubulin represents an evolutionary stage intermediate between prokaryotic FtsZ and eukaryotic microtubule-forming tubulins.
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Células Eucariotas , Tubulina (Proteína) , Eucariontes/metabolismo , Células Eucariotas/metabolismo , Guanosina Trifosfato/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/químicaRESUMEN
Pannexin (PANX) family proteins form large-pore channels that mediate purinergic signaling. We analyzed the cryo-EM structures of human PANX1 in lipid nanodiscs to elucidate the gating mechanism and its regulation by the amino terminus in phospholipids. The wild-type channel has an amino-terminal funnel in the pore, but in the presence of the inhibitor probenecid, a cytoplasmically oriented amino terminus and phospholipids obstruct the pore. Functional analysis using whole-cell patch-clamp and oocyte voltage clamp showed that PANX1 lacking the amino terminus did not open and had a dominant negative effect on channel activity, thus confirming that the amino-terminal domain played an essential role in channel opening. These observations suggest that dynamic conformational changes in the amino terminus of human PANX1 are associated with lipid movement in and out of the pore. Moreover, the data provide insight into the gating mechanism of PANX1 and, more broadly, other large-pore channels.
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Conexinas , Fosfolípidos , Conexinas/genética , Conexinas/metabolismo , Humanos , Proteínas del Tejido Nervioso/genética , Oocitos/metabolismo , Transducción de SeñalRESUMEN
ATP11C is a member of the P4-ATPase flippase family that mediates translocation of phosphatidylserine (PtdSer) across the lipid bilayer. In order to characterize the structure and function of ATP11C in a model natural lipid environment, we revisited and optimized a quick procedure for reconstituting ATP11C into Nanodiscs using methyl-ß-cyclodextrin as a reagent for the detergent removal. ATP11C was efficiently reconstituted with the endogenous lipid, or the mixture of endogenous lipid and synthetic dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylserine (DOPS), all of which retained the ATPase activity. We obtained 3.4 Å and 3.9 Å structures using single-particle cryo-electron microscopy (cryo-EM) of AlF- and BeF-stabilized ATP11C transport intermediates, respectively, in a bilayer containing DOPS. We show that the latter exhibited a distended inner membrane around ATP11C transmembrane helix 2, possibly reflecting the perturbation needed for phospholipid release to the lipid bilayer. Our structures of ATP11C in the lipid membrane indicate that the membrane boundary varies upon conformational changes of the enzyme and is no longer flat around the protein, a change that likely contributes to phospholipid translocation across the membrane leaflets.
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Adenosina Trifosfatasas , Membrana Dobles de Lípidos , Fosfolípidos , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismoRESUMEN
The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. Previous structural analysis of the K+-occluded E2-P transition state of H+,K+-ATPase showed a single bound K+ at cation-binding site II, in marked contrast to the two K+ ions occluded at sites I and II of the closely-related Na+,K+-ATPase which mediates electrogenic 3Na+/2K+ translocation across the membrane. The molecular basis of the different K+ stoichiometry between these K+-counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H+,K+-ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K+ bound H+,K+-ATPase, achieved by five mutations, including amino acids directly coordinating K+ (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K+-occluded conformation (Tyr799Trp). This quintuple mutant in the K+-occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K+ accommodated at their cation-binding site.
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Mucosa Gástrica/enzimología , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Potasio/metabolismo , Sitios de Unión/genética , Cationes Monovalentes/metabolismo , Membrana Celular/enzimología , Microscopía por Crioelectrón , Cristalización , Pruebas de Enzimas , Mucosa Gástrica/citología , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , ATPasa Intercambiadora de Hidrógeno-Potásio/aislamiento & purificación , ATPasa Intercambiadora de Hidrógeno-Potásio/ultraestructura , Células HEK293 , Humanos , Modelos Moleculares , Mutación , Ingeniería de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Especificidad por Sustrato/genéticaRESUMEN
The molecular shield effect was studied for intrinsically disordered proteins (IDPs) that do not adopt compact and stable protein folds. IDPs are found among many stress-responsive gene products and cryoprotective- and drought-protective proteins. We recently reported that some fragments of human genome-derived IDPs are cryoprotective for cellular enzymes, despite a lack of relevant amino acid sequence motifs. This sequence-independent IDP function may reflect their molecular shield effect. This study examined the inhibitory activity of IDPs against fibril formation in an amyloid beta peptide (Aß(1-42)) model system. Four of five human genome-derived IDPs (size range 20 to 44 amino acids) showed concentration-dependent inhibition of amyloid formation (IC50 range between 60 and 130 µM against 20 µM Aß(1-42)). The IC50 value was two orders of magnitude lower than that of polyethylene-glycol and dextran, used as neutral hydrophilic polymer controls. Nuclear magnetic resonance with 15 N-labeled Aß(1-42) revealed no relevant molecular interactions between Aß(1-42) and IDPs. The inhibitory activities were abolished by adding external amyloid-formation seeds. Therefore, IDPs seemed to act only at the amyloid nucleation phase but not at the elongation phase. These results suggest that IDPs (0.1 mM or less) have a molecular shield effect that prevents aggregation of susceptible molecules.
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Péptidos beta-Amiloides/química , Proteínas Intrínsecamente Desordenadas/química , Fragmentos de Péptidos/química , Péptidos beta-Amiloides/genética , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Fragmentos de Péptidos/genéticaRESUMEN
Regulating intercellular communication is essential for multicellular organisms. Gap junction channels are the major components mediating this function, but the molecular mechanisms underlying their opening and closing remain unclear. Single-particle cryo-electron microscopy (cryo-EM) is a powerful tool for investigating high-resolution protein structures that are difficult to crystallize, such as gap junction channels. Membrane protein structures are often determined in a detergent solubilized form, but lipid bilayers provide a near native environment for structural analysis. This review focuses on recent reports of gap junction channel structures visualized by cryo-EM. An overview of the differences observed in gap junction channel structures in the presence and absence of lipids is described, which may contribute to elucidating the regulation mechanisms of gap junction channel function.
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Microscopía por Crioelectrón , Uniones Comunicantes/química , Canales Iónicos/química , Modelos Moleculares , Animales , Conexinas/química , Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Canales Iónicos/metabolismo , Fosfolípidos/química , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Relación Estructura-ActividadRESUMEN
Gap junctions form intercellular conduits with a large pore size whose closed and open states regulate communication between adjacent cells. The structural basis of the mechanism by which gap junctions close, however, remains uncertain. Here, we show the cryo-electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction proteins in an undocked hemichannel form. In the nanodisc-reconstituted structure of the wild-type INX-6 hemichannel, flat double-layer densities obstruct the channel pore. Comparison of the hemichannel structures of a wild-type INX-6 in detergent and nanodisc-reconstituted amino-terminal deletion mutant reveals that lipid-mediated amino-terminal rearrangement and pore obstruction occur upon nanodisc reconstitution. Together with molecular dynamics simulations and electrophysiology functional assays, our results provide insight into the closure of the INX-6 hemichannel in a lipid bilayer before docking of two hemichannels.
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Proteínas de Caenorhabditis elegans/ultraestructura , Caenorhabditis elegans/metabolismo , Conexinas/ultraestructura , Microscopía por Crioelectrón , Simulación del Acoplamiento Molecular , Fosfolípidos/química , Animales , Proteínas de Caenorhabditis elegans/química , Conexinas/química , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Nanopartículas/química , Oocitos/metabolismo , Xenopus/metabolismoRESUMEN
Gap junction family proteins form conduits connecting the cytoplasm of adjacent cells, thereby enabling electrical and chemical coupling to maintain physiological homeostasis. Gap junction proteins comprise two gene families, connexins in chordates and innexins in pre-chordates. Their channel structures have been analyzed by electron or X-ray crystallography, but only a few atomic structures have been reported. Recent advances in single-particle cryo-electron microscopy (cryo-EM) will help to elucidate these structures further. Here the structural biology of gap junction channels utilizing crystallography and single-particle cryo-EM is overviewed to shed light on the functional mechanisms of cell-cell communication that are essential for multicellular organisms.
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Microscopía por Crioelectrón , Uniones Comunicantes/metabolismo , Animales , Conexinas/química , Conexinas/metabolismo , Cristalografía por Rayos X , HumanosRESUMEN
Gap junction channels are essential for mediating intercellular communication in most multicellular organisms. Two gene families encode gap junction channels, innexin and connexin. Although the sequence similarity between these two families based on bioinformatics is not conclusively determined, the gap junction channels encoded by these two gene families are structurally and functionally analogous. We recently reported an atomic structure of an invertebrate innexin gap junction channel using single-particle cryo-electron microscopy. Our findings revealed that connexin and innexin families share several structural properties with regard to their monomeric and oligomeric structures, while simultaneously suggesting a diversity of gap junction channels in nature. This review summarizes cutting-edge progress toward determining an innexin gap junction channel structure, as well as essential tips for preparing cryo-electron microscopy samples for high-resolution structural analysis of an innexin gap junction channel.
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Conexinas/ultraestructura , Microscopía por Crioelectrón/métodos , Uniones Comunicantes/ultraestructura , Manejo de Especímenes/métodos , Animales , Transporte Biológico , Comunicación Celular , Conexinas/química , Uniones Comunicantes/química , Manejo de Especímenes/instrumentaciónRESUMEN
Innexins, a large protein family comprising invertebrate gap junction channels, play an essential role in nervous system development and electrical synapse formation. Here we report the cryo-electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction channels at atomic resolution. We find that the arrangements of the transmembrane helices and extracellular loops of the INX-6 monomeric structure are highly similar to those of connexin-26 (Cx26), despite the lack of significant sequence similarity. The INX-6 gap junction channel comprises hexadecameric subunits but reveals the N-terminal pore funnel, consistent with Cx26. The helix-rich cytoplasmic loop and C-terminus are intercalated one-by-one through an octameric hemichannel, forming a dome-like entrance that interacts with N-terminal loops in the pore. These observations suggest that the INX-6 cytoplasmic domains are cooperatively associated with the N-terminal funnel conformation, and an essential linkage of the N-terminal with channel activity is presumably preserved across gap junction families.
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Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/ultraestructura , Caenorhabditis elegans/metabolismo , Conexinas/metabolismo , Conexinas/ultraestructura , Microscopía por Crioelectrón , Uniones Comunicantes/metabolismo , Uniones Comunicantes/ultraestructura , Animales , Proteínas de Caenorhabditis elegans/química , Conexinas/química , Modelos Moleculares , Dominios Proteicos , Homología Estructural de ProteínaRESUMEN
Innexins are invertebrate-specific gap junction proteins with four transmembrane helices. These proteins oligomerize to constitute intercellular channels that allow for the passage of small signaling molecules associated with neural and muscular electrical activity. In contrast to the large number of structural and functional studies of connexin gap junction channels, few structural studies of recombinant innexin channels are reported. Here we show the three-dimensional structure of two-dimensionally crystallized Caenorhabditis elegans innexin-6 (INX-6) gap junction channels. The N-terminal deleted INX-6 proteins are crystallized in lipid bilayers. The three-dimensional reconstruction determined by cryo-electron crystallography reveals that a single INX-6 gap junction channel comprises 16 subunits, a hexadecamer, in contrast to chordate connexin channels, which comprise 12 subunits. The channel pore diameters at the cytoplasmic entrance and extracellular gap region are larger than those of connexin26. Two bulb densities are observed in each hemichannel, one in the pore and the other at the cytoplasmic side of the hemichannel in the channel pore pathway. These findings imply a structural diversity of gap junction channels among multicellular organisms.
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Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Conexinas/química , Conexinas/metabolismo , Multimerización de Proteína , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , Uniones Comunicantes/química , Uniones Comunicantes/ultraestructura , Imagenología Tridimensional , Conformación ProteicaRESUMEN
We developed a method, named GraDeR, which substantially improves the preparation of membrane protein complexes for structure determination by single-particle cryo-electron microscopy (cryo-EM). In GraDeR, glycerol gradient centrifugation is used for the mild removal of free detergent monomers and micelles from lauryl maltose-neopentyl glycol detergent stabilized membrane complexes, resulting in monodisperse and stable complexes to which standard processes for water-soluble complexes can be applied. We demonstrate the applicability of the method on three different membrane complexes, including the mammalian FoF1 ATP synthase. For this highly dynamic and fragile rotary motor, we show that GraDeR allows visualizing the asymmetry of the F1 domain, which matches the ground state structure of the isolated domain. Therefore, the present cryo-EM structure of FoF1 ATP synthase provides direct structural evidence for Boyer's binding change mechanism in the context of the intact enzyme.