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Yorodumi- PDB-7qh2: Cryo-EM structure of Ldh-EtfAB complex from Acetobacterium woodii -
+Open data
-Basic information
Entry | Database: PDB / ID: 7qh2 | ||||||
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Title | Cryo-EM structure of Ldh-EtfAB complex from Acetobacterium woodii | ||||||
Components | (Lactate dehydrogenase (NAD(+),ferredoxin) subunit ...) x 3 | ||||||
Keywords | FLAVOPROTEIN / Electron bifurcation / Electron confirmation / Lactate / Lactate dehydrogenase complex / Electron transferring flavoprotein / A. woodii / redox enzyme | ||||||
Function / homology | Function and homology information lactate dehydrogenase (NAD+,ferredoxin) / D-lactate dehydrogenase activity / FAD binding / flavin adenine dinucleotide binding / 4 iron, 4 sulfur cluster binding / electron transfer activity / oxidoreductase activity / metal ion binding / cytoplasm Similarity search - Function | ||||||
Biological species | Acetobacterium woodii (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.43 Å | ||||||
Authors | Kayastha, K. / Ermler, U. | ||||||
Funding support | Germany, 1items
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Citation | Journal: Elife / Year: 2022 Title: Structure-based electron-confurcation mechanism of the Ldh-EtfAB complex. Authors: Kanwal Kayastha / Alexander Katsyv / Christina Himmrich / Sonja Welsch / Jan M Schuller / Ulrich Ermler / Volker Müller / Abstract: Lactate oxidation with NAD as electron acceptor is a highly endergonic reaction. Some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) ...Lactate oxidation with NAD as electron acceptor is a highly endergonic reaction. Some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) using a lactate dehydrogenase (Ldh) in concert with the electron-transferring proteins EtfA and EtfB. The electron cryo-microscopically characterized (Ldh-EtfAB) complex of at 2.43 Å resolution consists of a mobile EtfAB shuttle domain located between the rigid central Ldh and the peripheral EtfAB base units. The FADs of Ldh and the EtfAB shuttle domain contact each other thereby forming the D (dehydrogenation-connected) state. The intermediary Glu37 and Glu139 may harmonize the redox potentials between the FADs and the pyruvate/lactate pair crucial for FBEC. By integrating Alphafold2 calculations a plausible novel B (bifurcation-connected) state was obtained allowing electron transfer between the EtfAB base and shuttle FADs. Kinetic analysis of enzyme variants suggests a correlation between NAD binding site and D-to-B-state transition implicating a 75° rotation of the EtfAB shuttle domain. The FBEC inactivity when truncating the ferredoxin domain of EtfA substantiates its role as redox relay. Lactate oxidation in Ldh is assisted by the catalytic base His423 and a metal center. On this basis, a comprehensive catalytic mechanism of the FBEC process was proposed. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7qh2.cif.gz | 377.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7qh2.ent.gz | 305.3 KB | Display | PDB format |
PDBx/mmJSON format | 7qh2.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7qh2_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 7qh2_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 7qh2_validation.xml.gz | 66.4 KB | Display | |
Data in CIF | 7qh2_validation.cif.gz | 98.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/qh/7qh2 ftp://data.pdbj.org/pub/pdb/validation_reports/qh/7qh2 | HTTPS FTP |
-Related structure data
Related structure data | 13960MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Lactate dehydrogenase (NAD(+),ferredoxin) subunit ... , 3 types, 6 molecules ADBECF
#1: Protein | Mass: 46222.496 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Acetobacterium woodii (bacteria) / Strain: ATCC 29683 / DSM 1030 / JCM 2381 / KCTC 1655 / WB1 / Gene: lctC, etfA, Awo_c08720 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): del-iscR / References: UniProt: H6LBB1, EC: 1.3.1.110 #2: Protein | Mass: 29187.627 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Acetobacterium woodii (bacteria) / Strain: ATCC 29683 / DSM 1030 / JCM 2381 / KCTC 1655 / WB1 / Gene: lctB, etfB, Awo_c08710 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): del-iscR / References: UniProt: H6LBB0, EC: 1.3.1.110 #3: Protein | Mass: 51267.797 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Acetobacterium woodii (bacteria) / Strain: ATCC 29683 / DSM 1030 / JCM 2381 / KCTC 1655 / WB1 / Gene: lctD, glcD, Awo_c08730 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): del-iscR / References: UniProt: H6LBS1, EC: 1.3.1.110 |
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-Non-polymers , 3 types, 10 molecules
#4: Chemical | ChemComp-FAD / #5: Chemical | #6: Water | ChemComp-HOH / | |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Ldh-EtfAB complex / Type: COMPLEX Details: Complex composed of a dimer of a trimer of EtfA, EtfB and Ldh subunits making a hexameric oligomer state. Each of the subunits contain 1 FAD co-factor making total of 6 FADs. 2 Ldh subunits ...Details: Complex composed of a dimer of a trimer of EtfA, EtfB and Ldh subunits making a hexameric oligomer state. Each of the subunits contain 1 FAD co-factor making total of 6 FADs. 2 Ldh subunits contain 1 Fe molecule each. Entity ID: #1-#3 / Source: RECOMBINANT | |||||||||||||||
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Molecular weight | Value: 0.22 MDa / Experimental value: YES | |||||||||||||||
Source (natural) | Organism: Acetobacterium woodii (bacteria) | |||||||||||||||
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: del-iscR / Plasmid: pET21a | |||||||||||||||
Buffer solution | pH: 7.5 / Details: pH 7.5 | |||||||||||||||
Buffer component |
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Specimen | Conc.: 1.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Sample was cross linked with 1 mM of BS3 before grid preparation. | |||||||||||||||
Specimen support | Details: 45mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3 | |||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K Details: Blot force of +20 and blotting time of 4 seconds before plunging |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 6.52 sec. / Electron dose: 106.17 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 9788 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 30 eV |
Image scans | Width: 5760 / Height: 4092 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 5517853 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 674283 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL / Space: REAL |