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- PDB-7qh2: Cryo-EM structure of Ldh-EtfAB complex from Acetobacterium woodii -

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Basic information

Entry
Database: PDB / ID: 7qh2
TitleCryo-EM structure of Ldh-EtfAB complex from Acetobacterium woodii
Components(Lactate dehydrogenase (NAD(+),ferredoxin) subunit ...) x 3
KeywordsFLAVOPROTEIN / 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 / metal ion binding / cytoplasm
Similarity search - Function
Electron transfer flavoprotein, beta subunit / Electron transfer flavoprotein, alpha subunit, N-terminal / Electron transfer flavoprotein, beta subunit, N-terminal / Electron transfer flavoprotein alpha subunit/FixB / Electron transfer flavoprotein domain / Electron transfer flavoprotein, alpha/beta-subunit, N-terminal / Electron transfer flavoprotein, alpha subunit, C-terminal / Electron transfer flavoprotein FAD-binding domain / Electron transfer flavoprotein domain / FAD-linked oxidase, C-terminal ...Electron transfer flavoprotein, beta subunit / Electron transfer flavoprotein, alpha subunit, N-terminal / Electron transfer flavoprotein, beta subunit, N-terminal / Electron transfer flavoprotein alpha subunit/FixB / Electron transfer flavoprotein domain / Electron transfer flavoprotein, alpha/beta-subunit, N-terminal / Electron transfer flavoprotein, alpha subunit, C-terminal / Electron transfer flavoprotein FAD-binding domain / Electron transfer flavoprotein domain / FAD-linked oxidase, C-terminal / FAD linked oxidases, C-terminal domain / Vanillyl-alcohol oxidase, C-terminal subdomain 2 / FAD-linked oxidase-like, C-terminal / FAD linked oxidase, N-terminal / FAD binding domain / FAD-binding domain, PCMH-type / PCMH-type FAD-binding domain profile. / FAD-binding, type PCMH, subdomain 2 / FAD-binding, type PCMH-like superfamily / DHS-like NAD/FAD-binding domain superfamily / Rossmann-like alpha/beta/alpha sandwich fold
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / : / Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctB / Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctC / Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctD
Similarity search - Component
Biological speciesAcetobacterium woodii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.43 Å
AuthorsKayastha, K. / Ermler, U.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: 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
DepositionDec 10, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 29, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 6, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctC
B: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctB
C: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctD
D: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctC
E: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctB
F: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)258,18114
Polymers253,3566
Non-polymers4,8258
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area34220 Å2
ΔGint-197 kcal/mol
Surface area77930 Å2

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Components

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Lactate dehydrogenase (NAD(+),ferredoxin) subunit ... , 3 types, 6 molecules ADBECF

#1: Protein Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctC / Electron transfer flavoprotein large subunit / Electron transfer flavoprotein subunit alpha / EtfA ...Electron transfer flavoprotein large subunit / Electron transfer flavoprotein subunit alpha / EtfA / Lactate dehydrogenase-Etf complex subunit LctC


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, lactate dehydrogenase (NAD+,ferredoxin)
#2: Protein Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctB / Electron transfer flavoprotein small subunit / Electron transfer flavoprotein subunit beta / EtfB / ...Electron transfer flavoprotein small subunit / Electron transfer flavoprotein subunit beta / EtfB / Lactate dehydrogenase-Etf complex subunit LctB


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, lactate dehydrogenase (NAD+,ferredoxin)
#3: Protein Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctD


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, lactate dehydrogenase (NAD+,ferredoxin)

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Non-polymers , 3 types, 10 molecules

#4: Chemical
ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE / Flavin adenine dinucleotide


Mass: 785.550 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#5: Chemical ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 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
Molecular weightValue: 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 solutionpH: 7.5 / Details: pH 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2150 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 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 supportDetails: 45mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: 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

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 30 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1crYOLOparticle selectionGeneral neural network model used
2EPU2.9.0image acquisitionAutomated AFIS corrected image collection
4Gctf1.06CTF correction
7UCSF ChimeraX1.2model fitting
9Coot0.9model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5517853
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 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 buildingProtocol: AB INITIO MODEL / Space: REAL

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