Journal: Proc Natl Acad Sci U S A / Year: 2025 Title: A splendid molecular factory: De- and reconstruction of the mammalian respiratory chain. Authors: Lukas Rimle / Ben P Phillips / Isabela M Codo Costa Barra / Noëlle Arnold / Charlie Hennebert / Thomas Meier / Christoph von Ballmoos / Abstract: Mitochondrial respiratory complexes I to IV and the FF-ATP synthase (complex V) are large protein assemblies producing the universal cellular energy currency adenosine triphosphate (ATP). Individual ...Mitochondrial respiratory complexes I to IV and the FF-ATP synthase (complex V) are large protein assemblies producing the universal cellular energy currency adenosine triphosphate (ATP). Individual complexes have been extensively studied in vitro, but functional co-reconstitution of several mammalian complexes into proteoliposomes, in particular, the combination of a primary pump with the ATP synthase, is less well understood. Here, we present a generic and scalable strategy to purify mammalian respiratory complexes I, III and the ATP synthase from enriched mitochondria in enzymatically fully active form, and procedures to reassemble the complexes into liposomes. A robust functionality can be shown by in situ monitoring of ATP synthesis rates and by using selected inhibitors of the respiratory chain complexes. By inclusion of cytochrome oxidase, our procedures allowed us to reconstruct the entire mitochondrial respiratory chain (complexes I, III, IV, and V) in ubiquinone Q containing liposomes, demonstrating oxidative phosphorylation by nicotinamide adenine dinucleotide hydrogen driven ATP synthesis. The system was fully coupled at all levels and was used to probe cardiolipin as an essential component to activate the mammalian respiratory chain. Structural characterization using electron cryomicroscopy allowed us to resolve apo-state complex III and complex V at high and medium resolution, respectively, using in silico particle sorting, confirming the presence of all protein subunits and cofactors in native stoichiometry and conformation. The reported findings will facilitate future endeavors to characterize or modulate these key bioenergetic processes.
Model: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot time of 4s with blot force of -3.
Details
Monodisperse sample was purified un-tagged from bovine heart tissue and reconstituted into peptidiscs before freezing.
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Electron microscopy
Microscope
TFS GLACIOS
Temperature
Min: 80.0 K / Max: 90.5 K
Alignment procedure
Coma free - Residual tilt: 10.0 mrad
Specialist optics
Phase plate: OTHER / Energy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV / Details: Tuned using TFS Sherpa Software
Image recording
Film or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number real images: 3947 / Average exposure time: 8.0 sec. / Average electron dose: 40.0 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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Movie
Controller
Open data
Basic information
Map data
Sample
Keywords
Function and homology information
Bos taurus (domestic cattle)
Authors
Switzerland, 
United Kingdom, 2 items 
Citation
Structure visualization
Downloads & links
emd_17461.png
http://ftp.pdbj.org/pub/emdb/structures/EMD-17461
X (Sec.)
Y (Row.)
Z (Col.)
Sample components
Processing
Electron microscopy
FIELD EMISSION GUN
