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- EMDB-15452: Cryo-EM structure of the nitrogen-fixation associated NADH:ferred... -

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Basic information

Entry
Database: EMDB / ID: EMD-15452
TitleCryo-EM structure of the nitrogen-fixation associated NADH:ferredoxin oxidoreductase RNF from Azotobacter vinelandii
Map dataunsharpened map
Sample
  • Complex: Heptameric complex of NADH:ferredoxin oxidoreductase RNF
    • Protein or peptide: x 5 types
  • Protein or peptide: x 2 types
  • Ligand: x 5 types
Keywordsmetalloprotein / flavoprotein / electron transfer and Na+/H+ translocation / MEMBRANE PROTEIN
Function / homology
Function and homology information


Translocases / electron transport chain / transmembrane transport / FMN binding / 4 iron, 4 sulfur cluster binding / electron transfer activity / metal ion binding / plasma membrane
Similarity search - Function
RnfH protein / RnfH superfamily / RnfH family Ubiquitin / Ion-translocating oxidoreductase complex, RnfB/RsxB / Ion-translocating oxidoreductase complex, subunit RnfC/RsxC / Ion-translocating oxidoreductase complex subunit E / Ion-translocating oxidoreductase complex, subunit RnfA/RsxA / Ion-translocating oxidoreductase complex Rnf, subunit D / Electron transport complex, RnfB/RsxB, Proteobacteria / RnfC Barrel sandwich hybrid domain ...RnfH protein / RnfH superfamily / RnfH family Ubiquitin / Ion-translocating oxidoreductase complex, RnfB/RsxB / Ion-translocating oxidoreductase complex, subunit RnfC/RsxC / Ion-translocating oxidoreductase complex subunit E / Ion-translocating oxidoreductase complex, subunit RnfA/RsxA / Ion-translocating oxidoreductase complex Rnf, subunit D / Electron transport complex, RnfB/RsxB, Proteobacteria / RnfC Barrel sandwich hybrid domain / : / RnfC Barrel sandwich hybrid domain / Ion-translocating oxidoreductase complex, subunit RnfG/RsxG / 4Fe-4S domain / Putative Fe-S cluster / 4Fe-4S domain profile. / NqrDE/RnfAE / Ion-translocating oxidoreductase NqrB/RnfD / : / Rnf-Nqr subunit, membrane protein / NQR2, RnfD, RnfE family / 4Fe-4S dicluster domain / FMN-binding / FMN-binding domain / FMN_bind / Molybdopterin synthase/thiamin biosynthesis sulphur carrier, beta-grasp / 4Fe-4S dicluster domain / Soluble ligand binding domain / SLBB domain / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain superfamily / Respiratory-chain NADH dehydrogenase 51 Kd subunit / 4Fe-4S ferredoxin, iron-sulphur binding, conserved site / 4Fe-4S ferredoxin-type iron-sulfur binding region signature. / 4Fe-4S ferredoxin-type iron-sulfur binding domain profile. / 4Fe-4S ferredoxin-type, iron-sulphur binding domain
Similarity search - Domain/homology
Ion-translocating oxidoreductase complex subunit G / Ion-translocating oxidoreductase complex subunit D / Ion-translocating oxidoreductase complex subunit C / Ion-translocating oxidoreductase complex subunit B / Ion-translocating oxidoreductase complex subunit A / Protein RnfH / Ion-translocating oxidoreductase complex subunit E
Similarity search - Component
Biological speciesAzotobacter vinelandii DJ (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsZhang L / Einsle O
Funding supportEuropean Union, Germany, 4 items
OrganizationGrant numberCountry
European Research Council (ERC)310656European Union
German Research Foundation (DFG)CRC 1381, project ID 403222702 Germany
German Research Foundation (DFG)RTG 2202, project ID 46710898 Germany
German Research Foundation (DFG)grant INST 35/134-1 FUGG Germany
CitationJournal: Nat Chem Biol / Year: 2024
Title: Architecture of the RNF1 complex that drives biological nitrogen fixation.
Authors: Lin Zhang / Oliver Einsle /
Abstract: Biological nitrogen fixation requires substantial metabolic energy in form of ATP as well as low-potential electrons that must derive from central metabolism. During aerobic growth, the free-living ...Biological nitrogen fixation requires substantial metabolic energy in form of ATP as well as low-potential electrons that must derive from central metabolism. During aerobic growth, the free-living soil diazotroph Azotobacter vinelandii transfers electrons from the key metabolite NADH to the low-potential ferredoxin FdxA that serves as a direct electron donor to the dinitrogenase reductases. This process is mediated by the RNF complex that exploits the proton motive force over the cytoplasmic membrane to lower the midpoint potential of the transferred electron. Here we report the cryogenic electron microscopy structure of the nitrogenase-associated RNF complex of A. vinelandii, a seven-subunit membrane protein assembly that contains four flavin cofactors and six iron-sulfur centers. Its function requires the strict coupling of electron and proton transfer but also involves major conformational changes within the assembly that can be traced with a combination of electron microscopy and modeling.
History
DepositionJul 24, 2022-
Header (metadata) releaseNov 1, 2023-
Map releaseNov 1, 2023-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15452.png

Downloads & links

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Map

FileDownload / File: emd_15452.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 280 pix.
= 229.6 Å		0.82 Å/pix.
x 280 pix.
= 229.6 Å		0.82 Å/pix.
x 280 pix.
= 229.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.19443077 - 0.9367607
Average (Standard dev.)0.0019276759 (±0.02037143)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 229.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: sharpened map

Fileemd_15452_additional_1.map
Annotationsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halp map A

Fileemd_15452_half_map_1.map
Annotationhalp map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halp map B

Fileemd_15452_half_map_2.map
Annotationhalp map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Heptameric complex of NADH:ferredoxin oxidoreductase RNF

EntireName: Heptameric complex of NADH:ferredoxin oxidoreductase RNF
Components
  • Complex: Heptameric complex of NADH:ferredoxin oxidoreductase RNF
    • Protein or peptide: Ion-translocating oxidoreductase complex subunit A
    • Protein or peptide: Ion-translocating oxidoreductase complex subunit B
    • Protein or peptide: Ion-translocating oxidoreductase complex subunit C
    • Protein or peptide: Ion-translocating oxidoreductase complex subunit E
    • Protein or peptide: Protein RnfH
  • Protein or peptide: Ion-translocating oxidoreductase complex subunit D
  • Protein or peptide: Ion-translocating oxidoreductase complex subunit G
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: FLAVIN MONONUCLEOTIDE
  • Ligand: RIBOFLAVIN

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Supramolecule #1: Heptameric complex of NADH:ferredoxin oxidoreductase RNF

SupramoleculeName: Heptameric complex of NADH:ferredoxin oxidoreductase RNF
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3, #5, #7
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Molecular weightTheoretical: 188 KDa

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Macromolecule #1: Ion-translocating oxidoreductase complex subunit A

MacromoleculeName: Ion-translocating oxidoreductase complex subunit A / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Translocases
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria) / Strain: DJ / ATCC BAA-1303
Molecular weightTheoretical: 19.942131 KDa
Recombinant expressionOrganism: Azotobacter vinelandii DJ (bacteria)
SequenceString:
MEYVLFLLGT VLVHNVVLVG FLGLCPFMGV SSKLDPSIGL AVATTLVMGL GGASSWLLEH YVLLPLGIGF IRILAYIVVI AGMVQLIEM IIRKASPSLY RSLGIYLPLI TTNCAVLGVP LLSVREGHDL TMAVLFGLGS GLGFSLIMII FAGLRERLAL A NVPAAFSG PPIAFVTAGL LALAFMGFGG LI

UniProtKB: Ion-translocating oxidoreductase complex subunit A

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Macromolecule #2: Ion-translocating oxidoreductase complex subunit B

MacromoleculeName: Ion-translocating oxidoreductase complex subunit B / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Translocases
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria) / Strain: DJ / ATCC BAA-1303
Molecular weightTheoretical: 17.704572 KDa
Recombinant expressionOrganism: Azotobacter vinelandii DJ (bacteria)
SequenceString:
MIEATLALTV MGVLLGCGLG LAARKFAVTD ENPLIKEVSD LMPGSQCGQC GFPGCGAAAV AIVEGNASVT CCPPGGVGLA EKLAAILGV PLDASQVAAP MLARVEASQC IGCTRCYRAC PTDAIVGASG QVHVVLEDAC TGCGKCRDAC PEDCVLLIPQ E QTLDTWRW DKPAAA

UniProtKB: Ion-translocating oxidoreductase complex subunit B

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Macromolecule #3: Ion-translocating oxidoreductase complex subunit C

MacromoleculeName: Ion-translocating oxidoreductase complex subunit C / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: Translocases
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria) / Strain: DJ / ATCC BAA-1303
Molecular weightTheoretical: 52.234195 KDa
Recombinant expressionOrganism: Azotobacter vinelandii DJ (bacteria)
SequenceString: MYFNLSSIRG GVHPAAHKDL SAALPIGSLP LPPRLYLPLR QHAGAEALPM VAVGDKVLKG QLLAFPPTEV SAPVHAPTSG RIVAIGPVP APHPSGLTTT GIVLESDGED RWIDLDVSTD PFAEDPLVLA DRVAKAGIVG LGGAIFPAAV KLKQGTRHEI K TVLVNGSE ...String:
MYFNLSSIRG GVHPAAHKDL SAALPIGSLP LPPRLYLPLR QHAGAEALPM VAVGDKVLKG QLLAFPPTEV SAPVHAPTSG RIVAIGPVP APHPSGLTTT GIVLESDGED RWIDLDVSTD PFAEDPLVLA DRVAKAGIVG LGGAIFPAAV KLKQGTRHEI K TVLVNGSE CEPYLTCDDR IMRERAEAIV DGARLIQHIL RAYSVVIAIE DNKPEALAAM RAAAEHFGAI EVMAVPALYP MG SAKQLIQ AVTGREVPAG GRSTDVGVLV HNAGTVYAIQ QALRFGRPLI SRVVTVSGAC VKTPQNLDVL IGTPVQALID ACG GLSGDP QQLLLGGPMM GAVLPSTEVP VIKGATGLLA LARHELPNKD PAPCIRCASC VDACPMGLTP LDMALYARAD DYDG ASEYG LRDCILCGCC SYVCPSHIPL VHYFQYAKGQ QDERRSAARK SDYIKRQTEV RAARLAEEEA AKAAAKAAKE AAKAA KAAK TKAAKPSNEV ES

UniProtKB: Ion-translocating oxidoreductase complex subunit C

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Macromolecule #4: Ion-translocating oxidoreductase complex subunit D

MacromoleculeName: Ion-translocating oxidoreductase complex subunit D / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: Translocases
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria) / Strain: DJ / ATCC BAA-1303
Molecular weightTheoretical: 39.189016 KDa
Recombinant expressionOrganism: Azotobacter vinelandii DJ (bacteria)
SequenceString: MSTISVAAGP FAHDRSSVNR IMLDVCLALT PATLFGLVMF GWPAINLWLV TCVSALAIEA ACLRLLGQPM RRLLDGSALL TGWLLAISL PPWAPWWIGV GGSLFAIGIG KQLYGGIGQN PFNPAMLARV ALLIAFPLQM TTWALPHPLF SSSAPGFFDS L AITFAGAP ...String:
MSTISVAAGP FAHDRSSVNR IMLDVCLALT PATLFGLVMF GWPAINLWLV TCVSALAIEA ACLRLLGQPM RRLLDGSALL TGWLLAISL PPWAPWWIGV GGSLFAIGIG KQLYGGIGQN PFNPAMLARV ALLIAFPLQM TTWALPHPLF SSSAPGFFDS L AITFAGAP LADGMTGATA LGNLKTELTL NRTAQEILEG GFSTISALFG STPGSLGETS ELLLLVGGVW LVLRRIIHWE IP VAILASV FVMATLAYLI NPERYAGGLY QLTSGGLILC AFFIATDPVT SPISRVGRLI FGVGCGVLIY VIRTWGSFPE AAA FAVLFM NALTPLIDRY WRPRAYGRNV RGKPLVAAKW TSQVKEVDKV

UniProtKB: Ion-translocating oxidoreductase complex subunit D

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Macromolecule #5: Ion-translocating oxidoreductase complex subunit E

MacromoleculeName: Ion-translocating oxidoreductase complex subunit E / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO / EC number: Translocases
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria) / Strain: DJ / ATCC BAA-1303
Molecular weightTheoretical: 25.576328 KDa
Recombinant expressionOrganism: Azotobacter vinelandii DJ (bacteria)
SequenceString: MSHCGAPSVP EPEKKVPWQY FTSALWQYNV ALVQMLALCP TLAVTTTATN GLGMGLATTL VLVMTNALIS SMRHTISPEV RNPVMIGVI AGVVTLTDMA MNAWMHELYK VLGLFIALIV TNCAVLGRAE SFCLRNPVIP SILDGAGMGA GFTAVLVVIG G IREILGSG ...String:
MSHCGAPSVP EPEKKVPWQY FTSALWQYNV ALVQMLALCP TLAVTTTATN GLGMGLATTL VLVMTNALIS SMRHTISPEV RNPVMIGVI AGVVTLTDMA MNAWMHELYK VLGLFIALIV TNCAVLGRAE SFCLRNPVIP SILDGAGMGA GFTAVLVVIG G IREILGSG TLFSQASSLL GSHFKWMEIT VIPDFQGILL AILPPGAFIV LGFLLAAKRV IDRKRAERRQ QTHGELVVLQ

UniProtKB: Ion-translocating oxidoreductase complex subunit E

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Macromolecule #6: Ion-translocating oxidoreductase complex subunit G

MacromoleculeName: Ion-translocating oxidoreductase complex subunit G / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: Translocases
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria) / Strain: DJ / ATCC BAA-1303
Molecular weightTheoretical: 25.932709 KDa
Recombinant expressionOrganism: Azotobacter vinelandii DJ (bacteria)
SequenceString: MNDTTMTPAE ENAAPAEAAA GKPTLLARLE KWRPMVAYQG LSLGLVCAVV ALLLLTGNIM THGTIAEQQM QDRLATLREV LPQSLYDNN PLADSFKVQD AELGEVEVLP ARLQGKLTAV VFQGRNIGYG GPIEQMMSVD AQGKILGVRV LTHKETPGLA D KIEASRSD ...String:
MNDTTMTPAE ENAAPAEAAA GKPTLLARLE KWRPMVAYQG LSLGLVCAVV ALLLLTGNIM THGTIAEQQM QDRLATLREV LPQSLYDNN PLADSFKVQD AELGEVEVLP ARLQGKLTAV VFQGRNIGYG GPIEQMMSVD AQGKILGVRV LTHKETPGLA D KIEASRSD WIKVFDGLSL ENTALDKWKV KKDGGQFDQF AGATITPRAV VKTVLQGLQF QARHAEQLKA EWSHPQFEK

UniProtKB: Ion-translocating oxidoreductase complex subunit G

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Macromolecule #7: Protein RnfH

MacromoleculeName: Protein RnfH / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria) / Strain: DJ / ATCC BAA-1303
Molecular weightTheoretical: 9.644859 KDa
Recombinant expressionOrganism: Azotobacter vinelandii DJ (bacteria)
SequenceString:
MRVSVVYADP AKPLQLSCKV EDGCSVEQAI QQSGVLRCCP DIDLKKQKVG VFGKFVKLDS PLKDGDRIEI YQRVTRVDDD DDDDDD

UniProtKB: Protein RnfH

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Macromolecule #8: FE2/S2 (INORGANIC) CLUSTER

MacromoleculeName: FE2/S2 (INORGANIC) CLUSTER / type: ligand / ID: 8 / Number of copies: 1 / Formula: FES
Molecular weightTheoretical: 175.82 Da
Chemical component information

ChemComp-FES:
FE2/S2 (INORGANIC) CLUSTER

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Macromolecule #9: DODECYL-BETA-D-MALTOSIDE

MacromoleculeName: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 9 / Number of copies: 15 / Formula: LMT
Molecular weightTheoretical: 510.615 Da
Chemical component information

ChemComp-LMT:
DODECYL-BETA-D-MALTOSIDE / detergent*YM

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Macromolecule #10: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 10 / Number of copies: 4 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #11: FLAVIN MONONUCLEOTIDE

MacromoleculeName: FLAVIN MONONUCLEOTIDE / type: ligand / ID: 11 / Number of copies: 3 / Formula: FMN
Molecular weightTheoretical: 456.344 Da
Chemical component information

ChemComp-FMN:
FLAVIN MONONUCLEOTIDE

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Macromolecule #12: RIBOFLAVIN

MacromoleculeName: RIBOFLAVIN / type: ligand / ID: 12 / Number of copies: 1 / Formula: RBF
Molecular weightTheoretical: 376.364 Da
Chemical component information

ChemComp-RBF:
RIBOFLAVIN

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statecell

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 47.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 94807
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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