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- EMDB-44752: The consensus EM map of electron bifurcating Nfn-ABC complexed wi... -

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Basic information

Entry
Database: EMDB / ID: EMD-44752
TitleThe consensus EM map of electron bifurcating Nfn-ABC complexed with NAD from Caldicellulosiruptor saccharolyticus
Map data
Sample
  • Complex: Csac Nfn-ABC complex
KeywordsElectron bifurcating enzyme / Nfn-type BfuABC complex / FMN/B1/C1 bifurcation site / OXIDOREDUCTASE
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_44752.png

Downloads & links

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Map

FileDownload / File: emd_44752.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: 0.13
Minimum - Maximum-0.6935229 - 1.4059258
Average (Standard dev.)0.00062999636 (±0.03175983)
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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Mask #1

Fileemd_44752_msk_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_44752_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44752_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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

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Supramolecule #1: Csac Nfn-ABC complex

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

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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 reconstructionResolution.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)
FSC plot (resolution estimation)

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