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- EMDB-44751: Structure of electron bifurcating Nfn-ABC complexed with NAD from... -

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Basic information

Entry
Database: EMDB / ID: EMD-44751
TitleStructure of electron bifurcating Nfn-ABC complexed with NAD from Caldicellulosiruptor saccharolyticus
Map data
Sample
  • Complex: Csac Nfn-ABC complex
    • Protein or peptide: Molybdopterin oxidoreductase
    • Protein or peptide: NADH dehydrogenase (Quinone)
    • Protein or peptide: NADH dehydrogenase (Ubiquinone), 24 kDa subunit
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: FLAVIN MONONUCLEOTIDE
  • Ligand: ZINC ION
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
KeywordsElectron bifurcating enzyme / Nfn-type BfuABC complex / FMN/B1/C1 bifurcation site / OXIDOREDUCTASE
Function / homology
Function and homology information


NADH dehydrogenase (quinone) / 2 iron, 2 sulfur cluster binding / 4 iron, 4 sulfur cluster binding / oxidoreductase activity / metal ion binding
Similarity search - Function
NADP-reducing hydrogenase subunit HndA / Dihydroprymidine dehydrogenase domain II / Dihydroprymidine dehydrogenase domain II, 4Fe-4S cluster / Molybdopterin oxidoreductase, molybdopterin cofactor binding site / Prokaryotic molybdopterin oxidoreductases signature 1. / Soluble ligand binding domain / SLBB domain / Molybdopterin oxidoreductase Fe4S4 domain / Alpha-helical ferredoxin / Molybdopterin oxidoreductase Fe4S4 domain ...NADP-reducing hydrogenase subunit HndA / Dihydroprymidine dehydrogenase domain II / Dihydroprymidine dehydrogenase domain II, 4Fe-4S cluster / Molybdopterin oxidoreductase, molybdopterin cofactor binding site / Prokaryotic molybdopterin oxidoreductases signature 1. / Soluble ligand binding domain / SLBB domain / Molybdopterin oxidoreductase Fe4S4 domain / Alpha-helical ferredoxin / Molybdopterin oxidoreductase Fe4S4 domain / 4Fe-4S binding domain / 2Fe-2S iron-sulfur cluster binding domain / Respiratory-chain NADH dehydrogenase 24 Kd subunit signature. / NuoE domain / NADH-quinone oxidoreductase subunit E-like / FAD/NAD(P)-binding domain / NADH-quinone oxidoreductase subunit E, N-terminal / NADH-ubiquinone oxidoreductase 51kDa subunit, iron-sulphur binding domain / NADH-ubiquinone oxidoreductase 51kDa subunit, iron-sulphur binding domain superfamily / NADH-ubiquinone oxidoreductase-F iron-sulfur binding region / NADH-ubiquinone oxidoreductase-F iron-sulfur binding region / Thioredoxin-like [2Fe-2S] ferredoxin / Pyridine nucleotide-disulphide oxidoreductase / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain superfamily / Nuo51 FMN-binding domain / Molybdopterin oxidoreductase, 4Fe-4S domain / Prokaryotic molybdopterin oxidoreductases 4Fe-4S domain profile. / Molybdopterin oxidoreductase / Molybdopterin oxidoreductase / Beta-grasp domain superfamily / 4Fe-4S dicluster domain / 2Fe-2S ferredoxin-type iron-sulfur binding domain profile. / 2Fe-2S ferredoxin-type iron-sulfur binding domain / 2Fe-2S ferredoxin-like superfamily / 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 / FAD/NAD(P)-binding domain superfamily / Thioredoxin-like superfamily
Similarity search - Domain/homology
NADH dehydrogenase (Ubiquinone), 24 kDa subunit / NADH dehydrogenase (Quinone) / Molybdopterin oxidoreductase
Similarity search - Component
Biological speciesCaldicellulosiruptor saccharolyticus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsLi H
Funding support United States, 1 items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-SC0020085 United States
CitationJournal: Commun Biol / Year: 2025
Title: Cryo-EM reveals a composite flavobicluster electron bifurcation site in the Bfu family member NfnABC.
Authors: Hua Li / Gerrit J Schut / Xiang Feng / Michael W W Adams / Huilin Li /
Abstract: The BfuABC family is a diverse group of electron bifurcating enzymes that play key roles in anaerobic microbial metabolism. Previous studies have focused almost exclusively on the BfuABC-type ...The BfuABC family is a diverse group of electron bifurcating enzymes that play key roles in anaerobic microbial metabolism. Previous studies have focused almost exclusively on the BfuABC-type hydrogenases but the mechanism and site of electron bifurcation remain unknown. Herein we focus on the Caldicellulosiruptor saccharolyticus (Csac) NfnABC-type Bfu enzyme that catalyzes the oxidation of NADPH and simultaneous reduction of NAD and the redox protein ferredoxin (Fd). Cryo-EM structures determined with and without NAD and Fd reveal seven FeS clusters and one FAD in NfnA, one FeS cluster in NfnC, and three FeS clusters, two Zn ions, and one FMN in NfnB. The Zn ions take the place of FeS clusters previously proposed in other Bfu family members. Csac Nfn for the first time defines the minimum bifurcation site as a flavobicluster consisting of FMN, a [4Fe-4S] (B1) cluster and a [2Fe-2S] (C1) cluster. Binding of NAD to the FMN triggers a series of conformational changes, crucial to the bifurcation of two electron pairs derived from NADPH by the [B1-FMN-C1] flavobicluster into low and high potential electrons that reduce Fd and NAD, respectively. The structures lay the foundation for investigations of the proposed reaction cycle common to all Bfu enzymes.
History
DepositionMay 6, 2024-
Header (metadata) releaseMar 5, 2025-
Map releaseMar 5, 2025-
UpdateMar 5, 2025-
Current statusMar 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44751.png

Downloads & links

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Map

FileDownload / File: emd_44751.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 480 pix.
= 397.44 Å		0.83 Å/pix.
x 480 pix.
= 397.44 Å		0.83 Å/pix.
x 480 pix.
= 397.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.11
Minimum - Maximum-0.037108447 - 1.9399015
Average (Standard dev.)0.00997923 (±0.029156122)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 397.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Csac Nfn-ABC complex

EntireName: Csac Nfn-ABC complex
Components
  • Complex: Csac Nfn-ABC complex
    • Protein or peptide: Molybdopterin oxidoreductase
    • Protein or peptide: NADH dehydrogenase (Quinone)
    • Protein or peptide: NADH dehydrogenase (Ubiquinone), 24 kDa subunit
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: FLAVIN MONONUCLEOTIDE
  • Ligand: ZINC ION
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

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Supramolecule #1: Csac Nfn-ABC complex

SupramoleculeName: Csac Nfn-ABC complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Caldicellulosiruptor saccharolyticus (bacteria)
Molecular weightTheoretical: 760 KDa

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Macromolecule #1: Molybdopterin oxidoreductase

MacromoleculeName: Molybdopterin oxidoreductase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Caldicellulosiruptor saccharolyticus (bacteria)
Molecular weightTheoretical: 130.025906 KDa
Recombinant expressionOrganism: Caldicellulosiruptor (bacteria)
SequenceString: MRLVRVNIDN KEIFAEEGKT ILEVAHENNI EIPHLCYDKR LKPYGACGLC VVEIEGSPKL ARACSTYVTD KMVIKTDSPR VRNARKMAL ELLLSEHRGD CRPPCVLACP AHTDCQGYVG LIANGQFREA VALIKEQLPF PASIGRVCPH PCEEACRRNM V DQPIAIAE ...String:
MRLVRVNIDN KEIFAEEGKT ILEVAHENNI EIPHLCYDKR LKPYGACGLC VVEIEGSPKL ARACSTYVTD KMVIKTDSPR VRNARKMAL ELLLSEHRGD CRPPCVLACP AHTDCQGYVG LIANGQFREA VALIKEQLPF PASIGRVCPH PCEEACRRNM V DQPIAIAE LKRFVGDIDL LDDGYIPPIK PKTGKKVAIV GGGPAGLTCA FFLAKEGHDI VVYEAMPKAG GMLRYGIPEY RL PKGILDK EIELIEKMGV QIKTNMRLGV DISLEYLRKN YDAVFLAVGA WKSSTLGCPG DSAEGVIGGI EFLRKVSMNQ PVN LGQRVL VVGGGNTAMD AARTAIRLGA KEVTVLYRRT REEMPAEDIE VNEAEEEGVK FQFLVAPIEV ITDGGRVRAL KCQR MRLGD MDESGRRRPV PIEGAEVIFE ADTIISAIGQ KVRVEDVEGL ELTRHGTIKV DEGTYQTSLE GVFAGGDAVT GPKIA IEAI AQGKNAARVI DSYLRGKLEP IKEPYYVKQE DLTPEDFKDR ERKPRVPLKV ANAEERKNNF REITSTMTEK EAIAEA SRC LECGCMDYFE CQLYKYVNQY DVDPQRLSGY KHKRYEPQKH PFIERNPDKC ILCGLCIRVC EEVVGVCALG FVNRGFE TI VKPEFGLPLE ETSCISCGQC ADICPTGACI GKQPVAKQVP VNTVATKTVC TFCGMGCEML VETKGNLIFD VSPVQSNE G MLCAFGRFGI KYVNDKDRIL APLIKVNGEL SKTTFDQALI ETAKKLQAIR ASYGKDSIAI IASQRLTNEE ALLLTKLAQ KLDTTVIGSF DLRESVLDRI FGLNASTNSF DEIYSTDLIV AVGKVAENHA VMGAKLKKAV ELGAKLVTIN NGETRADERA IATYKIDNT AFFKATIKAL FEMKAVDEDY VSKIAVNLDE LKDDVKNVEV TDEASEFAKI IAGAKTAMVI VDEESVSDTT I GQLANILT LTQKIGRPRC GIIKVTGLGN TQGAWDMGIR MSKEGIVKLI NEGKVKAAFI VSEDPQAADK NLGEVLDKLE CL IVADVFL TETGKRADVV LPLVSHVEST GTVTRADGKI QNLNLVLKPK NGLSNLDLLL KLAELFGLQY NLEKLNREMV ELL QNENKY NQQILYTEGF ATPDKTVHLF VSKDAPAFVE KAVFDTVKNR FEKYLQDKHL KY

UniProtKB: Molybdopterin oxidoreductase

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Macromolecule #2: NADH dehydrogenase (Quinone)

MacromoleculeName: NADH dehydrogenase (Quinone) / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO / EC number: NADH dehydrogenase (quinone)
Source (natural)Organism: Caldicellulosiruptor saccharolyticus (bacteria)
Molecular weightTheoretical: 63.533527 KDa
Recombinant expressionOrganism: Caldicellulosiruptor (bacteria)
SequenceString: MKIRVGLGSC GMAAGGNKVM ECIQQELRSR NLDIPVEPTG CIGLCFFEPL VDVIDGDDVY TYGNVTPEMI PKIIESHVIG KKPLDEFIV STSFEPYPML KSQVRIALKN CGRINPEDID DYIKNGGYEA LKKVLTSMTP EEVIEEIKIS GLRGRGGAGF P TWFKWDAA ...String:
MKIRVGLGSC GMAAGGNKVM ECIQQELRSR NLDIPVEPTG CIGLCFFEPL VDVIDGDDVY TYGNVTPEMI PKIIESHVIG KKPLDEFIV STSFEPYPML KSQVRIALKN CGRINPEDID DYIKNGGYEA LKKVLTSMTP EEVIEEIKIS GLRGRGGAGF P TWFKWDAA RKASGDIKYV VCNADEGDPG AFMDRSILEG DPHAVLEGMT IAAYAIGAKE GYIYVRAEYP LAIKRLEIAI EQ ARNRNLL GNNILNTNFS FDIKLKKGAG AFVCGEETAL IASIEGERGM PRLKPPFPAQ SGLWGRPTNI NNVETYANVP WII TNGGKA FASLGTEKSK GTKVFALAGK IKRGGLVEVP MGMSLREVIY NIGGGIKDDK AFKAVQMGGP SGGCIPADLI DTPV DYESI TKTGAIMGSG GMIVMDETTC MVDIARFFLE FTCKESCGKC TYCRVGTRRM LEILDRICNG EGRDGDLELL EELAV SVKD GSLCGLGQTA PNPVLTTLRY FKDEYIAHIR DKKCPAKQCK ALITYSILPE KCTGCGLCAR KCPTKAITGE RLKPHV IDQ SKCTKCGTCM NVCRFGAVNV E

UniProtKB: NADH dehydrogenase (Quinone)

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Macromolecule #3: NADH dehydrogenase (Ubiquinone), 24 kDa subunit

MacromoleculeName: NADH dehydrogenase (Ubiquinone), 24 kDa subunit / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Caldicellulosiruptor saccharolyticus (bacteria)
Molecular weightTheoretical: 19.914066 KDa
Recombinant expressionOrganism: Caldicellulosiruptor (bacteria)
SequenceString:
MYMSCPECEN RLASNFKNQK VDLSLLDPVL DEYKGEKSNI IAILQKTQEI YRFLPLDALN YISEKTGVKK AKIYGIATFY AQFRLKPVG KYVILQCQGT ACHVNGSEEI KNALCDELNI KPGDTTEDGM FTLEEVACLG CCSLAPVMMI NGETYGKLTP D KAREIIRR IYEREKNV

UniProtKB: NADH dehydrogenase (Ubiquinone), 24 kDa subunit

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Macromolecule #4: FE2/S2 (INORGANIC) CLUSTER

MacromoleculeName: FE2/S2 (INORGANIC) CLUSTER / type: ligand / ID: 4 / Number of copies: 4 / Formula: FES
Molecular weightTheoretical: 175.82 Da
Chemical component information

ChemComp-FES:
FE2/S2 (INORGANIC) CLUSTER

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Macromolecule #5: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 5 / Number of copies: 28 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #6: FLAVIN-ADENINE DINUCLEOTIDE

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

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

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Macromolecule #7: FLAVIN MONONUCLEOTIDE

MacromoleculeName: FLAVIN MONONUCLEOTIDE / type: ligand / ID: 7 / Number of copies: 4 / Formula: FMN
Molecular weightTheoretical: 456.344 Da
Chemical component information

ChemComp-FMN:
FLAVIN MONONUCLEOTIDE

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #9: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 9 / Number of copies: 4 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM

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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
Component:
ConcentrationFormulaName
260.0 mMNaClSodium Chloride
25.0 mMHEPES
1.0 1%trehalose
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 302 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 193.0 K / Max: 193.0 K
Alignment procedureComa free - Residual tilt: 0.05 mrad
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 16457 / Average exposure time: 1.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 1.0 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: 463896
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1.0) / Number images used: 78470
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.1.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.1.0)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.1.0)

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