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Institute
The TOM complex is the main entry point for precursor proteins into mitochondria. Precursor proteins containing targeting sequences are recognized by the TOM complex and imported into the mitochondria. We have determined the structure of the TOM core complex from Neurospora crassa by single-particle cryoEM at 3.3 Å resolution, showing its interaction with a bound presequence at 4 Å resolution, and of the TOM holo complex including the Tom20 receptor at 6-7 Å resolution. TOM is a transmembrane complex consisting of two β-barrels, three receptor subunits and three short transmembrane subunits. Tom20 has a transmembrane helix and a receptor domain on the cytoplasmic side. We propose that Tom20 acts as a dynamic gatekeeper, guiding precursor proteins into the pores of the TOM complex. We analyze the interactions of Tom20 with other TOM subunits, present insights into the structure of the TOM holo complex, and suggest a translocation mechanism.
The major light-harvesting complex (LHC-II) of higher plants plays a crucial role in capturing light energy for photosynthesis and in regulating the flow of energy within the photosynthetic apparatus. Native LHC-II isolated from plant tissue consists of three isoforms, Lhcb1, Lhcb2, and Lhcb3, which form homo- and heterotrimers. All three isoforms are highly conserved among different species, suggesting distinct functional roles. We produced the three LHC-II isoforms by heterologous expression of the polypeptide in Escherichia coli and in vitro refolding with purified pigments. Although Lhcb1 and Lhcb2 are very similar in polypeptide sequence and pigment content, Lhcb3 is clearly different because it lacks an N-terminal phosphorylation site and has a higher chlorophyll a/b ratio, suggesting the absence of one chlorophyll b. Low temperature absorption and fluorescence emission spectra of the pure isoforms revealed small but significant differences in pigment organization. The oligomeric state of the pure isoforms and of their permutations was investigated by native gel electrophoresis, sucrose density gradient centrifugation, and SDS-PAGE. Lhcb1 and Lhcb2 formed trimeric complexes by themselves and with one another, but Lhcb3 was able to do so only in combination with one or both of the other isoforms. We conclude that the main role of Lhcb1 and Lhcb2 is in the adaptation of photosynthesis to different light regimes. The most likely role of Lhcb3 is as an intermediary in light energy transfer from the main Lhcb1/Lhcb2 antenna to the photosystem II core.
Single-particle electron cryo-microscopy (cryoEM) has undergone a “resolution revolution” that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions or sample preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. We report a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, our new protocol has yielded a 3.1 Å map of yeast FAS from 15,000 automatically picked particles within a day. The high map quality enabled us to build a complete atomic model of an intact fungal FAS.
Single-particle electron cryo-microscopy (cryoEM) has undergone a `resolution revolution' that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions and sample-preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. Here, a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM is reported. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, the new protocol yielded a 3.1 Å resolution map of yeast FAS from 15 000 automatically picked particles within a day. The high map quality enabled a complete atomic model of an intact fungal FAS to be built.
β-barrel proteins mediate nutrient uptake in bacteria and serve vital functions in cell signaling and adhesion. For the 14-strand outer membrane protein G of Escherichia coli, opening and closing is pH-dependent. Different roles of the extracellular loops in this process were proposed, and X-ray and solution NMR studies were divergent. Here, we report the structure of outer membrane protein G investigated in bilayers of E. coli lipid extracts by magic-angle-spinning NMR. In total, 1847 inter-residue 1H–1H and 13C–13C distance restraints, 256 torsion angles, but no hydrogen bond restraints are used to calculate the structure. The length of β-strands is found to vary beyond the membrane boundary, with strands 6–8 being the longest and the extracellular loops 3 and 4 well ordered. The site of barrel closure at strands 1 and 14 is more disordered than most remaining strands, with the flexibility decreasing toward loops 3 and 4. Loop 4 presents a well-defined helix.
Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by ∼30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus.
Highlights
• Cryo-EM structure of a yeast F1Fo-ATP synthase dimer
• Inhibitor-free X-ray structure of the F1 head and rotor complex
• Mechanism of ATP generation by rotary catalysis
• Structural basis of cristae formation in the inner mitochondrial membrane
Summary
We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing.
Electron transfer in respiratory chains generates the electrochemical potential that serves as energy source for the cell. Prokaryotes can use a wide range of electron donors and acceptors and may have alternative complexes performing the same catalytic reactions as the mitochondrial complexes. This is the case for the alternative complex III (ACIII), a quinol:cytochrome c/HiPIP oxidoreductase. In order to understand the catalytic mechanism of this respiratory enzyme, we determined the structure of ACIII from Rhodothermus marinus at 3.9 Å resolution by single-particle cryo-electron microscopy. ACIII presents a so-far unique structure, for which we establish the arrangement of the cofactors (four iron–sulfur clusters and six c-type hemes) and propose the location of the quinol-binding site and the presence of two putative proton pathways in the membrane. Altogether, this structure provides insights into a mechanism for energy transduction and introduces ACIII as a redox-driven proton pump.
Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions face the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.
We examined substrate-induced conformational changes in MjNhaP1, an archaeal electroneutral Na+/H+-antiporter resembling the human antiporter NHE1, by electron crystallography of 2D crystals in a range of physiological pH and Na+ conditions. In the absence of sodium, changes in pH had no major effect. By contrast, changes in Na+ concentration caused a marked conformational change that was largely pH-independent. Crystallographically determined, apparent dissociation constants indicated ∼10-fold stronger Na+ binding at pH 8 than at pH 4, consistent with substrate competition for a common ion-binding site. Projection difference maps indicated helix movements by about 2 Å in the 6-helix bundle region of MjNhaP1 that is thought to contain the ion translocation site. We propose that these movements convert the antiporter from the proton-bound, outward-open state to the Na+-bound, inward-open state. Oscillation between the two states would result in rapid Na+/H+ antiport.