Complementary structures of the yeast phosphate transporter Pho90 provide insights into its transport mechanism.

Phosphate homeostasis is essential for all living organisms. Low-affinity phosphate transporters are involved in phosphate import and regulation in a range of eukaryotic organisms. We have determined the structures of the Saccharomyces cerevisiae phosphate importer Pho90 by electron cryomicroscopy in two complementary states at 2.3 and 3.1 Å resolution. The symmetrical, outward-open structure in the presence of phosphate indicates bound substrate ions in the binding pocket. In the absence of phosphate, Pho90 assumes an asymmetric structure with one monomer facing inward and one monomer facing outward, providing insights into the transport mechanism. The Pho90 transport domain binds phosphate ions on one side of the membrane, then flips to the other side where the substrate is released. Together with functional experiments, these complementary structures illustrate the transport mechanism of eukaryotic low-affinity phosphate transporters.

Two conformations of the Tom20 preprotein receptor in the TOM holo complex.

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

Analysis of the conformational heterogeneity of the Rieske iron-sulfur protein in complex III2 by cryo-EM.

Movement of the Rieske domain of the iron-sulfur protein is essential for intramolecular electron transfer within complex III2 (CIII2) of the respiratory chain as it bridges a gap in the cofactor chain towards the electron acceptor cytochrome c. We present cryo-EM structures of CIII2 from Yarrowia lipolytica at resolutions up to 2.0 Šunder different conditions, with different redox states of the cofactors of the high-potential chain. All possible permutations of three primary positions were observed, indicating that the two halves of the dimeric complex act independently. Addition of the substrate analogue decylubiquinone to CIII2 with a reduced high-potential chain increased the occupancy of the Qo site. The extent of Rieske domain interactions through hydrogen bonds to the cytochrome b and cytochrome c1 subunits varied depending on the redox state and substrate. In the absence of quinols, the reduced Rieske domain interacted more closely with cytochrome b and cytochrome c1 than in the oxidized state. Upon addition of the inhibitor antimycin A, the heterogeneity of the cd1-helix and ef-loop increased, which may be indicative of a long-range effect on the Rieske domain.

Cryo-EM structure of the respiratory I + III2 supercomplex from Arabidopsis thaliana at 2 Å resolution.

Protein complexes of the mitochondrial respiratory chain assemble into respiratory supercomplexes. Here we present the high-resolution electron cryo-microscopy structure of the Arabidopsis respiratory supercomplex consisting of complex I and a complex III dimer, with a total of 68 protein subunits and numerous bound cofactors. A complex I-ferredoxin, subunit B14.7 and P9, a newly defined subunit of plant complex I, mediate supercomplex formation. The component complexes stabilize one another, enabling new detailed insights into their structure. We describe (1) an interrupted aqueous passage for proton translocation in the membrane arm of complex I; (2) a new coenzyme A within the carbonic anhydrase module of plant complex I defining a second catalytic centre; and (3) the water structure at the proton exit pathway of complex III2 with a co-purified ubiquinone in the QO site. We propose that the main role of the plant supercomplex is to stabilize its components in the membrane.

Conformational changes in mitochondrial complex I of the thermophilic eukaryote Chaetomium thermophilum.

Mitochondrial complex I is a redox-driven proton pump that generates proton-motive force across the inner mitochondrial membrane, powering oxidative phosphorylation and ATP synthesis in eukaryotes. We report the structure of complex I from the thermophilic fungus Chaetomium thermophilum, determined by cryoEM up to 2.4-Å resolution. We show that the complex undergoes a transition between two conformations, which we refer to as state 1 and state 2. The conformational switch is manifest in a twisting movement of the peripheral arm relative to the membrane arm, but most notably in substantial rearrangements of the Q-binding cavity and the E-channel, resulting in a continuous aqueous passage from the E-channel to subunit ND5 at the far end of the membrane arm. The conformational changes in the complex interior resemble those reported for mammalian complex I, suggesting a highly conserved, universal mechanism of coupling electron transport to proton pumping.

Insights into complex I assembly: Function of NDUFAF1 and a link with cardiolipin remodeling.

Respiratory complex I is a ~1-MDa proton pump in mitochondria. Its structure has been revealed in great detail, but the structural basis of its assembly, in humans involving at least 15 assembly factors, is essentially unknown. We determined cryo-electron microscopy structures of assembly intermediates associated with assembly factor NDUFAF1 in a yeast model system. Subunits ND2 and NDUFC2 together with assembly factors NDUFAF1 and CIA84 form the nucleation point of the NDUFAF1-dependent assembly pathway. Unexpectedly, the cardiolipin remodeling enzyme tafazzin is an integral component of this core complex. In a later intermediate, all 12 subunits of the proximal proton pump module have assembled. NDUFAF1 locks the central ND3 subunit in an assembly-competent conformation, and major rearrangements of central subunits are required for complex I maturation.

Ion transfer mechanisms in Mrp-type antiporters from high resolution cryoEM and molecular dynamics simulations.

Multiple resistance and pH adaptation (Mrp) cation/proton antiporters are essential for growth of a variety of halophilic and alkaliphilic bacteria under stress conditions. Mrp-type antiporters are closely related to the membrane domain of respiratory complex I. We determined the structure of the Mrp antiporter from Bacillus pseudofirmus by electron cryo-microscopy at 2.2 Å resolution. The structure resolves more than 99% of the sidechains of the seven membrane subunits MrpA to MrpG plus 360 water molecules, including ~70 in putative ion translocation pathways. Molecular dynamics simulations based on the high-resolution structure revealed details of the antiport mechanism. We find that switching the position of a histidine residue between three hydrated pathways in the MrpA subunit is critical for proton transfer that drives gated trans-membrane sodium translocation. Several lines of evidence indicate that the same histidine-switch mechanism operates in respiratory complex I.

Concluding remarks: Challenges and future developments in biological electron cryo-microscopy.

During the past 10 years, biological electron cryo-microscopy (cryoEM) has undergone a process of rapid transformation. Many things we could only dream about a decade ago have now become almost routine. Nevertheless, a number of challenges remain, to do with sample preparation, the correlation between tomographic analysis and light microscopy, data validation, and the growing impact of artificial intelligence and structure prediction. This year's Faraday Discussion examined these challenges in some detail. The concluding remarks present a concise summary of the meeting and a brief outlook to the future.

Ca2+-mediated higher-order assembly of heterodimers in amino acid transport system b0,+ biogenesis and cystinuria.

Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, causing recurrent kidney stones and kidney failure. Mutations of the regulatory glycoprotein rBAT and the amino acid transporter b0,+AT, which constitute system b0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Here, using electron cryo-microscopy and biochemistry, we discover that Ca2+ mediates higher-order assembly of system b0,+. Ca2+ stabilizes the interface between two rBAT molecules, leading to super-dimerization of b0,+AT-rBAT, which in turn facilitates N-glycan maturation and protein trafficking. A cystinuria mutant T216M and mutations of the Ca2+ site of rBAT cause the loss of higher-order assemblies, resulting in protein trapping at the ER and the loss of function. These results provide the molecular basis of system b0,+ biogenesis and type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and protein-trafficking diseases.

Forty years in cryoEM of membrane proteins.

In a surprisingly short time, electron cryo-microscopy (cryoEM) has developed from a niche technique in structural biology to a mainstream method practiced in a rapidly growing number of laboratories around the world. From its beginnings about 40 years ago, cryoEM has had a major impact on the study of membrane proteins, in particular the energy-converting systems from bacterial, mitochondrial and chloroplast membranes. Early work on two-dimensional crystals attained resolutions ∼3.5 Å, but at present, single-particle cryoEM delivers much more detailed structures without crystals. Electron cryo-tomography of membranes and membrane-associated proteins adds valuable context, usually at lower resolution. The review ends with a brief outlook on future prospects.

Conformational changes in the yeast mitochondrial ABC transporter Atm1 during the transport cycle.

The mitochondrial inner membrane ABC transporter Atm1 exports an unknown substrate to the cytosol for iron-sulfur protein biogenesis, cellular iron regulation, and tRNA thio-modification. Mutations in the human relative ABCB7 cause the iron storage disease XLSA/A. We determined 3D structures of two complementary states of Atm1 in lipid nanodiscs by electron cryo-microscopy at 2.9- to 3.4-Å resolution. The inward-open structure resembled the known crystal structure of nucleotide-free apo-Atm1 closely. The occluded conformation with bound AMP-PNP-Mg2+ showed a tight association of the two nucleotide-binding domains, a rearrangement of the C-terminal helices, and closure of the putative substrate-binding cavity in the homodimeric transporter. We identified a hydrophobic patch on the C-terminal helices of yeast Atm1, which is unique among type IV ABC transporters of known structure. Truncation mutants of yeast Atm1 suggest that the C-terminal helices stabilize the dimer, yet are not necessary for closure of the nucleotide-binding domains.

High-resolution structure and dynamics of mitochondrial complex I-Insights into the proton pumping mechanism.

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a 1-MDa membrane protein complex with a central role in energy metabolism. Redox-driven proton translocation by complex I contributes substantially to the proton motive force that drives ATP synthase. Several structures of complex I from bacteria and mitochondria have been determined, but its catalytic mechanism has remained controversial. We here present the cryo-EM structure of complex I from Yarrowia lipolytica at 2.1-Å resolution, which reveals the positions of more than 1600 protein-bound water molecules, of which ~100 are located in putative proton translocation pathways. Another structure of the same complex under steady-state activity conditions at 3.4-Å resolution indicates conformational transitions that we associate with proton injection into the central hydrophilic axis. By combining high-resolution structural data with site-directed mutagenesis and large-scale molecular dynamic simulations, we define details of the proton translocation pathways and offer insights into the redox-coupled proton pumping mechanism of complex I.

Structure of the hexameric fungal plasma membrane proton pump in its autoinhibited state.

The fungal plasma membrane H+-ATPase Pma1 is a vital enzyme, generating a proton-motive force that drives the import of essential nutrients. Autoinhibited Pma1 hexamers in the plasma membrane of starving fungi are activated by glucose signaling and subsequent phosphorylation of the autoinhibitory domain. As related P-type adenosine triphosphatases (ATPases) are not known to oligomerize, the physiological relevance of Pma1 hexamers remained unknown. We have determined the structure of hexameric Pma1 from Neurospora crassa by electron cryo-microscopy at 3.3-Å resolution, elucidating the molecular basis for hexamer formation and autoinhibition and providing a basis for structure-based drug development. Coarse-grained molecular dynamics simulations in a lipid bilayer suggest lipid-mediated contacts between monomers and a substantial protein-induced membrane deformation that could act as a proton-attracting funnel.

Molecular landscape of etioplast inner membranes in higher plants.

Etioplasts are photosynthetically inactive plastids that accumulate when light levels are too low for chloroplast maturation. The etioplast inner membrane consists of a paracrystalline tubular lattice and peripheral, disk-shaped membranes, respectively known as the prolamellar body and prothylakoids. These distinct membrane regions are connected into one continuous compartment. To date, no structures of protein complexes in or at etioplast membranes have been reported. Here, we used electron cryo-tomography to explore the molecular membrane landscape of pea and maize etioplasts. Our tomographic reconstructions show that ATP synthase monomers are enriched in the prothylakoids, and plastid ribosomes in the tubular lattice. The entire tubular lattice is covered by regular helical arrays of a membrane-associated protein, which we identified as the 37-kDa enzyme, light-dependent protochlorophyllide oxidoreductase (LPOR). LPOR is the most abundant protein in the etioplast, where it is responsible for chlorophyll biosynthesis, photoprotection and defining the membrane geometry of the prolamellar body. Based on the 9-Å-resolution volume of the subtomogram average, we propose a structural model of membrane-associated LPOR.

A ferredoxin bridge connects the two arms of plant mitochondrial complex I.

Mitochondrial complex I is the main site for electron transfer to the respiratory chain and generates much of the proton gradient across the inner mitochondrial membrane. Complex I is composed of two arms, which form a conserved L-shape. We report the structures of the intact, 47-subunit mitochondrial complex I from Arabidopsis thaliana and the 51-subunit complex I from the green alga Polytomella sp., both at around 2.9 Šresolution. In both complexes, a heterotrimeric γ-carbonic anhydrase domain is attached to the membrane arm on the matrix side. Two states are resolved in A. thaliana complex I, with different angles between the two arms and different conformations of the ND1 (NADH dehydrogenase subunit 1) loop near the quinol binding site. The angle appears to depend on a bridge domain, which links the peripheral arm to the membrane arm and includes an unusual ferredoxin. We propose that the bridge domain participates in regulating the activity of plant complex I.

Current limitations to high-resolution structure determination by single-particle cryoEM.

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