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- EMDB-13960: Cryo-EM structure of Ldh-EtfAB complex from Acetobacterium woodii -

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Basic information

Entry
Database: EMDB / ID: EMD-13960
TitleCryo-EM structure of Ldh-EtfAB complex from Acetobacterium woodii
Map data
Sample
  • Complex: Ldh-EtfAB complex
    • Protein or peptide: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctC
    • Protein or peptide: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctB
    • Protein or peptide: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctD
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: FE (III) ION
  • Ligand: water
KeywordsElectron bifurcation / Electron confirmation / Lactate / Lactate dehydrogenase complex / Electron transferring flavoprotein / A. woodii / redox enzyme / FLAVOPROTEIN
Function / homology
Function and homology information


lactate dehydrogenase (NAD+,ferredoxin) / D-lactate dehydrogenase (NAD+) activity / fatty acid beta-oxidation using acyl-CoA dehydrogenase / FAD binding / flavin adenine dinucleotide binding / 4 iron, 4 sulfur cluster binding / oxidoreductase activity / 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 domain / Electron transfer flavoprotein alpha subunit/FixB / 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 ...: / Electron transfer flavoprotein, beta subunit / Electron transfer flavoprotein, alpha subunit, N-terminal / Electron transfer flavoprotein, beta subunit, N-terminal / Electron transfer flavoprotein domain / Electron transfer flavoprotein alpha subunit/FixB / 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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.43 Å
AuthorsKayastha K / Ermler U
Funding support Germany, 1 items
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, 2021-
Header (metadata) releaseJun 29, 2022-
Map releaseJun 29, 2022-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13960.png

Downloads & links

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Map

FileDownload / File: emd_13960.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 288 pix.
= 267.84 Å		0.93 Å/pix.
x 288 pix.
= 267.84 Å		0.93 Å/pix.
x 288 pix.
= 267.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0102
Minimum - Maximum-0.035595242 - 0.13561597
Average (Standard dev.)0.000084687585 (±0.0026789645)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 267.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13960_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ldh-EtfAB complex

EntireName: Ldh-EtfAB complex
Components
  • Complex: Ldh-EtfAB complex
    • Protein or peptide: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctC
    • Protein or peptide: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctB
    • Protein or peptide: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctD
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: FE (III) ION
  • Ligand: water

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Supramolecule #1: Ldh-EtfAB complex

SupramoleculeName: Ldh-EtfAB complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
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.
Source (natural)Organism: Acetobacterium woodii (bacteria)
Molecular weightTheoretical: 220 KDa

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Macromolecule #1: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctC

MacromoleculeName: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctC
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: ec: 1.3.1.110
Source (natural)Organism: Acetobacterium woodii (bacteria) / Strain: ATCC 29683 / DSM 1030 / JCM 2381 / KCTC 1655 / WB1
Molecular weightTheoretical: 46.222496 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAGIKIIKEN VDRETFEALA EICPFDAFSY ENDKLEVTAA CKMCKMCLKK GPEGVLILEE DEKVAIDKSL YRGITVYVDH IEGQIHPVT FELIGKAREL AAVIGHPVYA LLMGTNITEK ADELLKYGVD KVFVYDKPEL KHFVIEPYAN VLEDFIEKVK P SSILVGAT ...String:
MAGIKIIKEN VDRETFEALA EICPFDAFSY ENDKLEVTAA CKMCKMCLKK GPEGVLILEE DEKVAIDKSL YRGITVYVDH IEGQIHPVT FELIGKAREL AAVIGHPVYA LLMGTNITEK ADELLKYGVD KVFVYDKPEL KHFVIEPYAN VLEDFIEKVK P SSILVGAT NVGRSLAPRV AARYRTGLTA DCTILEMKEN TDLVQIRPAF GGNIMAQIVT ENTRPQFCTV RYKVFTAPER VN EPWGDVE MMDIEKAKLV SAIEVMEVIK KEKGIDLSEA ETIVAVGRGV KCEKDLDMIH EFAEKIGATV ACTRPGIEAG WFD ARLQIG LSGRTVKPKL IIALGISGAV QFAAGMQNSE YIIAINSDPK APIFNIAHCG MVGDLYEILP ELLTMIEGPE NNKD TETIS IPEAIETPER MVV

UniProtKB: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctC

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Macromolecule #2: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctB

MacromoleculeName: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctB
type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: ec: 1.3.1.110
Source (natural)Organism: Acetobacterium woodii (bacteria) / Strain: ATCC 29683 / DSM 1030 / JCM 2381 / KCTC 1655 / WB1
Molecular weightTheoretical: 29.187627 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSKILVCIKQ VPGTSNVEVD PETGVLIRDG VESKLNPYDL FGLETAFRLK EQLGGTITTL SMGPMQSKEV LMESFYMGAD EGCLLSDRK FGGADVVATS YTLAQGTKRL GDFDLIICGK QTTDGDTAQV GPEMAEFLGI PHVTNVIKIL AADEKGLTLQ M NMEESLEI ...String:
MSKILVCIKQ VPGTSNVEVD PETGVLIRDG VESKLNPYDL FGLETAFRLK EQLGGTITTL SMGPMQSKEV LMESFYMGAD EGCLLSDRK FGGADVVATS YTLAQGTKRL GDFDLIICGK QTTDGDTAQV GPEMAEFLGI PHVTNVIKIL AADEKGLTLQ M NMEESLEI QRVPYPCLIT VDKDIYTPRL PSYKRKLDIS KNPEIKILTL KDMYDTNEKK YGLSGSPTQV ERIFPPESNV EK TSFEGDG KVLAKALLGI LTEKKYLG

UniProtKB: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctB

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

MacromoleculeName: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctD
type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: ec: 1.3.1.110
Source (natural)Organism: Acetobacterium woodii (bacteria) / Strain: ATCC 29683 / DSM 1030 / JCM 2381 / KCTC 1655 / WB1
Molecular weightTheoretical: 51.267797 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNYKKVEASD IAAIKELIPA ERVFVGTEIG EDFSHDELGS IHSYPEVLIK VTSTEEVSKI MKYAYEHNIP VVVRGSGTGL VGACVPLFG GIMLETTLMN NILELDTENL TVTVEPGVLL MELSKFVEEN DLFYPPDPGE KSATIAGNIS TNAGGMRAVK Y GVTRDYVR ...String:
MNYKKVEASD IAAIKELIPA ERVFVGTEIG EDFSHDELGS IHSYPEVLIK VTSTEEVSKI MKYAYEHNIP VVVRGSGTGL VGACVPLFG GIMLETTLMN NILELDTENL TVTVEPGVLL MELSKFVEEN DLFYPPDPGE KSATIAGNIS TNAGGMRAVK Y GVTRDYVR GLTVVLANGE IIELGGKIVK NSSGYSLKDL VIGSEGTLCV ITKAILKLLP LPKMTLSLLI PFENISDAAG IV PKIIKSK AIPTAIEFME RQTILFAEDF LGKKFPDSSS NAYILLTFDG NTKEQVEAEY ETVANLCLAE GAKDVYIVDT VER KDSVWS ARGAFLEAIK ASTTEMDECD VVVPRNRIAE FIEFTHDLAK EMDVRIPSFG HAGDGNLHIY VCRDELCQAD WEAK LAEAM DRMYAKALTF EGLVSGEHGI GYAKRKYLLN DFGTEHLALM AGIKQTFDPK NLLNPKKVCQ MA

UniProtKB: Lactate dehydrogenase (NAD(+),ferredoxin) subunit LctD

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Macromolecule #4: FLAVIN-ADENINE DINUCLEOTIDE

MacromoleculeName: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 4 / Number of copies: 6 / Formula: FAD
Molecular weightTheoretical: 785.55 Da
Chemical component information

ChemComp-FAD:
FLAVIN-ADENINE DINUCLEOTIDE / FAD*YM

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Macromolecule #5: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 2 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHEPES
150.0 mMNaClSodium Chloride

Details: pH 7.5
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 135 sec. / Pretreatment - Atmosphere: NITROGEN / Pretreatment - Pressure: 38.0 kPa / Details: 45mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: Blot force of +20 and blotting time of 4 seconds before plunging.
DetailsSample was cross linked with 1 mM of BS3 before grid preparation.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 30 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number real images: 9788 / Average exposure time: 6.52 sec. / Average electron dose: 106.17 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5517853
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.43 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 674283
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 500000 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

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

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