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- EMDB-14622: Na+ - translocating ferredoxin: NAD+ reductase (Rnf) of C. tetano... -

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Basic information

Entry
Database: EMDB / ID: EMD-14622
TitleNa+ - translocating ferredoxin: NAD+ reductase (Rnf) of C. tetanomorphum
Map data
Sample
  • Complex: Na+ -translocating ferredoxin: NAD+ reductase (Rnf)
    • Protein or peptide: RnfA
    • Protein or peptide: RnfB
    • Protein or peptide: RnfC
    • Protein or peptide: RnfD
    • Protein or peptide: RnfE
    • Protein or peptide: RnfG
  • Ligand: FE (III) ION
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster
  • Ligand: FLAVIN MONONUCLEOTIDE
  • Ligand: RIBOFLAVIN
Biological speciesClostridium tetanomorphum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.27 Å
AuthorsErmler U / Vitt S / Buckel W
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)VI 778/2-1 Germany
CitationJournal: Nat Commun / Year: 2022
Title: Purification and structural characterization of the Na-translocating ferredoxin: NAD reductase (Rnf) complex of Clostridium tetanomorphum.
Authors: Stella Vitt / Simone Prinz / Martin Eisinger / Ulrich Ermler / Wolfgang Buckel /
Abstract: Various microbial metabolisms use H/Na-translocating ferredoxin:NAD reductase (Rnf) either to exergonically oxidize reduced ferredoxin by NAD for generating a transmembrane electrochemical potential ...Various microbial metabolisms use H/Na-translocating ferredoxin:NAD reductase (Rnf) either to exergonically oxidize reduced ferredoxin by NAD for generating a transmembrane electrochemical potential or reversely to exploit the latter for producing reduced ferredoxin. For cryo-EM structural analysis, we elaborated a quick four-step purification protocol for the Rnf complex from Clostridium tetanomorphum and integrated the homogeneous and active enzyme into a nanodisc. The obtained 4.27 Å density map largely allows chain tracing and redox cofactor identification complemented by biochemical data from entire Rnf and single subunits RnfB, RnfC and RnfG. On this basis, we postulated an electron transfer route between ferredoxin and NAD via eight [4Fe-4S] clusters, one Fe ion and four flavins crossing the cell membrane twice related to the pathway of NADH:ubiquinone reductase. Redox-coupled Na translocation is provided by orchestrating Na uptake/release, electrostatic effects of the assumed membrane-integrated FMN semiquinone anion and accompanied polypeptide rearrangements mediated by different redox steps.
History
DepositionMar 25, 2022-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateNov 23, 2022-
Current statusNov 23, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14622.png

Downloads & links

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Map

FileDownload / File: emd_14622.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.837 Å
Density
Contour LevelBy AUTHOR: 0.0115
Minimum - Maximum-0.049992055 - 0.14030054
Average (Standard dev.)2.7384343e-05 (±0.0027154044)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 200.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14622_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_14622_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_14622_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Na+ -translocating ferredoxin: NAD+ reductase (Rnf)

EntireName: Na+ -translocating ferredoxin: NAD+ reductase (Rnf)
Components
  • Complex: Na+ -translocating ferredoxin: NAD+ reductase (Rnf)
    • Protein or peptide: RnfA
    • Protein or peptide: RnfB
    • Protein or peptide: RnfC
    • Protein or peptide: RnfD
    • Protein or peptide: RnfE
    • Protein or peptide: RnfG
  • Ligand: FE (III) ION
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster
  • Ligand: FLAVIN MONONUCLEOTIDE
  • Ligand: RIBOFLAVIN

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Supramolecule #1: Na+ -translocating ferredoxin: NAD+ reductase (Rnf)

SupramoleculeName: Na+ -translocating ferredoxin: NAD+ reductase (Rnf) / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Clostridium tetanomorphum (bacteria)
Molecular weightTheoretical: 165 KDa

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Macromolecule #1: RnfA

MacromoleculeName: RnfA / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium tetanomorphum (bacteria)
Molecular weightTheoretical: 20.59485 KDa
Recombinant expressionOrganism: Clostridium tetanomorphum (bacteria)
SequenceString:
MSIFTIFISA LLVNNFVLSR FLGICPFLGV SKKVETATGM GAAVTFVMAL AAIMTFLVER FILIPLNIQY LSTLAFILVI ASLVQFVEM VIKKVSPDLY KALGIYLPLI TTNCAVLGMA VINSNEKYNL IQSIINSVGA ALGFTLALVL LAGIREKMET N EYIPEALK GLPITLVTAG LMAIAFLGFQ GLI

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Macromolecule #2: RnfB

MacromoleculeName: RnfB / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium tetanomorphum (bacteria)
Molecular weightTheoretical: 28.008584 KDa
Recombinant expressionOrganism: Clostridium tetanomorphum (bacteria)
SequenceString: MEGLLFPVLS LGGLGVVFGL LLGYASKKFA VEVDERVPMV RAALPGANCG GCGFAGCDAY ADAVVNAGAK PNGCPVGGAA CAAKIAEIM GVVVDSSEPK KAYVKCQGTC DKAKEKYEYY GAMTCVDAAN IPGAGSKTCG FGCLGLGSCV QVCAFDAIHV E NGIAVVDE ...String:
MEGLLFPVLS LGGLGVVFGL LLGYASKKFA VEVDERVPMV RAALPGANCG GCGFAGCDAY ADAVVNAGAK PNGCPVGGAA CAAKIAEIM GVVVDSSEPK KAYVKCQGTC DKAKEKYEYY GAMTCVDAAN IPGAGSKTCG FGCLGLGSCV QVCAFDAIHV E NGIAVVDE EACTGCGACV SICPKSVIEL TPMSKKVRIS CNSHDKGIEV KNACSVGCLS CGLCVRNCPS EAITMVNNLP VI DYDKCTQ CGVCVGKCPT KAIVNLNTNV SKEASNN

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Macromolecule #3: RnfC

MacromoleculeName: RnfC / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium tetanomorphum (bacteria)
Molecular weightTheoretical: 47.164734 KDa
Recombinant expressionOrganism: Clostridium tetanomorphum (bacteria)
SequenceString: MELLTFKNGV HPPHGKHYTE NKPIEEYLPK GDIVIPMSQH IGAPAEPIVK KGDRVLVGQK IGEAKGFVSA NIHASVSGTV KNVAPVTLF NGVKSTAVII ENDGQYEEIE TEKRDYTKLS NEEIINIIKE AGIVGMGGAT FPTHVKLAPP PDKNIDSIVV N AAECEPYL ...String:
MELLTFKNGV HPPHGKHYTE NKPIEEYLPK GDIVIPMSQH IGAPAEPIVK KGDRVLVGQK IGEAKGFVSA NIHASVSGTV KNVAPVTLF NGVKSTAVII ENDGQYEEIE TEKRDYTKLS NEEIINIIKE AGIVGMGGAT FPTHVKLAPP PDKNIDSIVV N AAECEPYL TCDHRMMLEK TNEIVEGLKI VLKLFPKATG YIGIEDNKMN AIKAMQEAVK NIANIEVKAV KTKYPQGAEK QL IYAITKR EVPSGGLPAD AGCIVQNVDT IYEIYNAVVN GKPLTSRVVT VTGDAIKEPK NLRFKIGTSV RELVEAAGGF AEE PLKVIS GGPMMGMAMY SLDVPSTKGT SGVLCLTKKV AEIEEESNCI NCGKCVQVCP MNLMPTKLAT ASAVSNLDMF NEFS GRDCI ECGCCSFVCP ARRHLLQRIR SGKKAVSKKK

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Macromolecule #4: RnfD

MacromoleculeName: RnfD / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium tetanomorphum (bacteria)
Molecular weightTheoretical: 32.990891 KDa
Recombinant expressionOrganism: Clostridium tetanomorphum (bacteria)
SequenceString: MSETTMYTVS SSPHIRAKDT TQSIMRDVVI ALLPATIAGV YFFKLQGLLV ILASVLSCVV AEYIWQKASK KKVTVGDYSA VVTGLLLAF NVPASIPLWI PVVGGFFAII VVKQFFGGLG QNIVNPALAA RAFLLASWPV QMTSWTLDGV TTATPLAILK G NEATGAAA ...String:
MSETTMYTVS SSPHIRAKDT TQSIMRDVVI ALLPATIAGV YFFKLQGLLV ILASVLSCVV AEYIWQKASK KKVTVGDYSA VVTGLLLAF NVPASIPLWI PVVGGFFAII VVKQFFGGLG QNIVNPALAA RAFLLASWPV QMTSWTLDGV TTATPLAILK G NEATGAAA PDLMSVFIGH VGGCIGETSA LALLIGGAYL FYKHIIDWRI PVSFIGTTFI FTAIAGRGSS PVYELFAGGL ML GAIFMAT DYATSPITPL GRIIFGVGCG VITSLIRIFG GYPEGVSYSI LVMNLFVPLI ERWTAPKIFG KVK

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Macromolecule #5: RnfE

MacromoleculeName: RnfE / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium tetanomorphum (bacteria)
Molecular weightTheoretical: 21.213398 KDa
Recombinant expressionOrganism: Clostridium tetanomorphum (bacteria)
SequenceString: MGVVSERLYN GIVKENATFV QVLGMCPTLA VTTSAINGIG MGLSATVVLI GSNVVISLLK KVIPDEIRIP AYITVIATLV TVLQFLLQA YLPDLNKSLG IFIPLIVVNC IILGRAEAYA NKNSVGASFF DGLGMGLGFT VSLAALGIIR EFLGTGKVFG A QITPDAFQ ...String:
MGVVSERLYN GIVKENATFV QVLGMCPTLA VTTSAINGIG MGLSATVVLI GSNVVISLLK KVIPDEIRIP AYITVIATLV TVLQFLLQA YLPDLNKSLG IFIPLIVVNC IILGRAEAYA NKNSVGASFF DGLGMGLGFT VSLAALGIIR EFLGTGKVFG A QITPDAFQ PALIMILAPG GFFTLGILMA ILNQRKLKKA KAK

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Macromolecule #6: RnfG

MacromoleculeName: RnfG / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium tetanomorphum (bacteria)
Molecular weightTheoretical: 19.502664 KDa
Recombinant expressionOrganism: Clostridium tetanomorphum (bacteria)
SequenceString:
MKKVSSFKLG MVLLLIAAVC GLILGGVNQV TAEPIAIQNK KTLDEANKAI LPEASEFAEK TDIKGEGIVL GVTEGKSGSD LKGYTIKVA PKGYAGAIEM MVGVSTEGKV TGIKILNHAE TPGLGANATD PKFSGQYANK PAKELKVVKG AASGEDEIVA I TGATITSK AVTLGVNEAI KFYDTKLKGG K

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Macromolecule #7: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #8: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 8 / Number of copies: 7 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER / Iron–sulfur cluster

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Macromolecule #9: FLAVIN MONONUCLEOTIDE

MacromoleculeName: FLAVIN MONONUCLEOTIDE / type: ligand / ID: 9 / Number of copies: 3 / Formula: FMN
Molecular weightTheoretical: 456.344 Da
Chemical component information

ChemComp-FMN:
FLAVIN MONONUCLEOTIDE / Flavin mononucleotide

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Macromolecule #10: RIBOFLAVIN

MacromoleculeName: RIBOFLAVIN / type: ligand / ID: 10 / Number of copies: 1 / Formula: RBF
Molecular weightTheoretical: 376.364 Da
Chemical component information

ChemComp-RBF:
RIBOFLAVIN / Riboflavin

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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

BufferpH: 7.5
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.27 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 88423
FSC plot (resolution estimation)

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