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- PDB-9bp5: Structure of electron bifurcating Nfn-ABC holoenzyme from Caldice... -

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Basic information

Entry
Database: PDB / ID: 9bp5
TitleStructure of electron bifurcating Nfn-ABC holoenzyme from Caldicellulosiruptor saccharolyticus
Components
  • (NADH dehydrogenase ...) x 2
  • Molybdopterin oxidoreductase
KeywordsOXIDOREDUCTASE / Electron bifurcating enzyme / Nfn-type BfuABC complex / FMN/B1/C1 bifurcation site
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 / Molybdopterin oxidoreductase Fe4S4 domain / Alpha-helical ferredoxin ...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 / Molybdopterin oxidoreductase Fe4S4 domain / Alpha-helical ferredoxin / 4Fe-4S binding domain / 2Fe-2S iron-sulfur cluster binding domain / Respiratory-chain NADH dehydrogenase 24 Kd subunit signature. / NuoE domain / FAD/NAD(P)-binding domain / NADH-quinone oxidoreductase subunit E-like / NADH-quinone oxidoreductase subunit E, N-terminal / Pyridine nucleotide-disulphide oxidoreductase / 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 / 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
FLAVIN-ADENINE DINUCLEOTIDE / FE2/S2 (INORGANIC) CLUSTER / FLAVIN MONONUCLEOTIDE / IRON/SULFUR CLUSTER / NADH dehydrogenase (Ubiquinone), 24 kDa subunit / NADH dehydrogenase (Quinone) / Molybdopterin oxidoreductase
Similarity search - Component
Biological speciesCaldicellulosiruptor saccharolyticus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsLi, H. / Li, H.
Funding support United States, 1items
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 7, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Molybdopterin oxidoreductase
B: NADH dehydrogenase (Quinone)
C: NADH dehydrogenase (Ubiquinone), 24 kDa subunit
D: Molybdopterin oxidoreductase
E: NADH dehydrogenase (Quinone)
F: NADH dehydrogenase (Ubiquinone), 24 kDa subunit
G: Molybdopterin oxidoreductase
H: NADH dehydrogenase (Quinone)
I: NADH dehydrogenase (Ubiquinone), 24 kDa subunit
J: Molybdopterin oxidoreductase
K: NADH dehydrogenase (Quinone)
L: NADH dehydrogenase (Ubiquinone), 24 kDa subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)873,45072
Polymers853,89412
Non-polymers19,55660
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 , 1 types, 4 molecules ADGJ

#1: Protein
Molybdopterin oxidoreductase


Mass: 130025.906 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caldicellulosiruptor saccharolyticus (bacteria)
Gene: Csac_0621 / Production host: Caldicellulosiruptor (bacteria) / References: UniProt: A4XH60

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NADH dehydrogenase ... , 2 types, 8 molecules BEHKCFIL

#2: Protein
NADH dehydrogenase (Quinone)


Mass: 63533.527 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caldicellulosiruptor saccharolyticus (bacteria)
Gene: Csac_0620 / Production host: Caldicellulosiruptor (bacteria) / References: UniProt: A4XH59, NADH dehydrogenase (quinone)
#3: Protein
NADH dehydrogenase (Ubiquinone), 24 kDa subunit


Mass: 19914.066 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caldicellulosiruptor saccharolyticus (bacteria)
Gene: Csac_0619 / Production host: Caldicellulosiruptor (bacteria) / References: UniProt: A4XH58

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Non-polymers , 5 types, 60 molecules

#4: Chemical
ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER


Mass: 175.820 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Fe2S2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical...
ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 36 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#7: Chemical
ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 8 / 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: Csac Nfn-ABC complex / Type: COMPLEX / Entity ID: #1-#3 / Source: NATURAL
Molecular weightValue: 0.76 MDa / Experimental value: YES
Source (natural)Organism: Caldicellulosiruptor saccharolyticus (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1260 mMSodium ChlorideNaCl1
225 mMHEPES1
31 1%trehalose1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 302 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 193 K / Temperature (min): 193 K / Residual tilt: 0.05 mradians
Image recordingAverage exposure time: 1 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7290
Image scansWidth: 5760 / Height: 4092

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Processing

EM softwareName: PHENIX / Version: 1.21rc1_4903: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 279815
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 108700 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00460736
ELECTRON MICROSCOPYf_angle_d0.49182292
ELECTRON MICROSCOPYf_dihedral_angle_d5.918356
ELECTRON MICROSCOPYf_chiral_restr0.0449416
ELECTRON MICROSCOPYf_plane_restr0.00410524

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