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- PDB-9n5u: Structure of the Thermococcus sibiricus NfnABC complex -

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Basic information

Entry
Database: PDB / ID: 9n5u
TitleStructure of the Thermococcus sibiricus NfnABC complex
Components
  • NfnA
  • NfnB
  • NfnC
KeywordsELECTRON TRANSPORT / Flavin-based electron bifurcation / Bfu family / Nfn-ABC / cryo-EM structures / flavo-FeS cluster
Function / homology
Function and homology information


iron-sulfur cluster binding / NADH dehydrogenase (ubiquinone) activity / ATP synthesis coupled electron transport / 2 iron, 2 sulfur cluster binding / 4 iron, 4 sulfur cluster binding / oxidoreductase activity / metal ion binding / membrane
Similarity search - Function
NADP-reducing hydrogenase subunit HndA / Dihydroprymidine dehydrogenase domain II / Dihydroprymidine dehydrogenase domain II, 4Fe-4S cluster / 4Fe-4S dicluster domain / Soluble ligand binding domain / SLBB domain / Alpha-helical ferredoxin / : / NADH-quinone oxidoreductase subunit 3, ferredoxin-like domain / Respiratory-chain NADH dehydrogenase 75 Kd subunit signature 1. ...NADP-reducing hydrogenase subunit HndA / Dihydroprymidine dehydrogenase domain II / Dihydroprymidine dehydrogenase domain II, 4Fe-4S cluster / 4Fe-4S dicluster domain / Soluble ligand binding domain / SLBB domain / Alpha-helical ferredoxin / : / NADH-quinone oxidoreductase subunit 3, ferredoxin-like domain / Respiratory-chain NADH dehydrogenase 75 Kd subunit signature 1. / NADH:ubiquinone oxidoreductase, 75kDa subunit, conserved site / 2Fe-2S iron-sulfur cluster binding domain / 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 / 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
FLAVIN-ADENINE DINUCLEOTIDE / FE2/S2 (INORGANIC) CLUSTER / FLAVIN MONONUCLEOTIDE / IRON/SULFUR CLUSTER / NADH:ubiquinone oxidoreductase, NADH-binding subunit F / NADH:ubiquinone oxidoreductase, subunit E / Glutamate synthase beta chain-related oxidoreductase, containing 2Fe-2S and 4Fe-4S clusters
Similarity search - Component
Biological speciesThermococcus sibiricus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsXiao, X. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
Department of Energy (DOE, United States) United States
CitationJournal: J Biol Chem / Year: 2025
Title: Cryo-EM structures define the electron bifurcating flavobicluster and ferredoxin binding site in an archaeal Nfn-Bfu transhydrogenase.
Authors: Xiansha Xiao / Gerrit J Schut / Xiang Feng / Diep M N Nguyen / Haiyan Huang / Shuning Wang / Huilin Li / Michael W W Adams /
Abstract: Flavin-based electron bifurcation couples exergonic and endergonic redox reactions in one enzyme complex to circumvent thermodynamic barriers and minimize free energy loss. Two unrelated enzymes ...Flavin-based electron bifurcation couples exergonic and endergonic redox reactions in one enzyme complex to circumvent thermodynamic barriers and minimize free energy loss. Two unrelated enzymes designated NfnSL and NfnABC catalyze the NADPH-dependent reduction of ferredoxin and NAD. Bifurcation by NfnSL resides with a single FAD but the bifurcation mechanism of NfnABC, which represents the diverse and ubiquitous Bfu enzyme family, is completely different and largely unknown. Using cryo-EM structures of an archaeal NfnABC, we show that its bifurcation site is a flavobicluster consisting of FMN, one [4Fe-4S] and one [2Fe-2S] cluster where zinc atoms replace two additional clusters previously identified in other Bfu enzymes. NADH binds to the flavobicluster site of NfnABC and induces conformational changes that allow ferredoxin to bind between the C-terminal domains of NfnC and NfnB. Site-directed mutational analyses support the proposed mechanism that is likely conserved in all members of the Bfu enzyme family.
History
DepositionFeb 4, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 7, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: NfnA
B: NfnB
C: NfnC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)196,07017
Polymers191,5333
Non-polymers4,53714
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein NfnA


Mass: 107912.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus sibiricus (archaea) / Gene: TSIB_1517 / Production host: Pyrococcus furiosus (archaea) / References: UniProt: C6A4M3
#2: Protein NfnB / NADH:ubiquinone oxidoreductase / NADH-binding subunit F


Mass: 66237.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus sibiricus (archaea) / Gene: XD54_1074 / Production host: Pyrococcus furiosus (archaea) / References: UniProt: A0A117L1A0
#3: Protein NfnC / NADH:ubiquinone oxidoreductase / subunit E


Mass: 17382.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus sibiricus (archaea) / Gene: XD54_1073 / Production host: Pyrococcus furiosus (archaea) / References: UniProt: A0A117L1U2

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Non-polymers , 5 types, 14 molecules

#4: Chemical
ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#6: Chemical ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER


Mass: 175.820 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: Fe2S2 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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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: Ternary complex of the Tsi NfnABC complex / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Thermococcus sibiricus (archaea)
Source (recombinant)Organism: Pyrococcus furiosus (archaea)
Buffer solutionpH: 7.5
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 18072
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 30

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2SerialEM4image acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 14943071
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 198898 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 2.85 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00513646
ELECTRON MICROSCOPYf_angle_d0.55818503
ELECTRON MICROSCOPYf_dihedral_angle_d11.6465131
ELECTRON MICROSCOPYf_chiral_restr0.0452069
ELECTRON MICROSCOPYf_plane_restr0.0052339

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