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- PDB-8xcn: Cryo-EM Structure of Membrane-bound Fructose Dehydrogenase from G... -

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Basic information

Entry
Database: PDB / ID: 8xcn
TitleCryo-EM Structure of Membrane-bound Fructose Dehydrogenase from Gluconobacter japonicus variant-N1190A
Components(Fructose dehydrogenase ...) x 3
KeywordsOXIDOREDUCTASE / Complex / OXIDOREDUCTASE Membrane-bound protein / OXIDEREDUCTASE
Function / homology
Function and homology information


fructose 5-dehydrogenase / fructose 5-dehydrogenase activity / oxidoreductase activity, acting on CH-OH group of donors / fructose metabolic process / flavin adenine dinucleotide binding / electron transfer activity / iron ion binding / heme binding / plasma membrane
Similarity search - Function
FAD-containing D-sorbitol dehydrogenase small subunit / Membrane bound FAD containing D-sorbitol dehydrogenase / Membrane-bound alcohol dehydrogenase, cytochrome c subunit / NAD(P)-binding Rossmann-like domain / Glucose-methanol-choline oxidoreductase, N-terminal / GMC oxidoreductase / Glucose-methanol-choline oxidoreductase, C-terminal / GMC oxidoreductase / Cytochrome c / Cytochrome c family profile. ...FAD-containing D-sorbitol dehydrogenase small subunit / Membrane bound FAD containing D-sorbitol dehydrogenase / Membrane-bound alcohol dehydrogenase, cytochrome c subunit / NAD(P)-binding Rossmann-like domain / Glucose-methanol-choline oxidoreductase, N-terminal / GMC oxidoreductase / Glucose-methanol-choline oxidoreductase, C-terminal / GMC oxidoreductase / Cytochrome c / Cytochrome c family profile. / Cytochrome c-like domain / Cytochrome c-like domain superfamily / FAD/NAD(P)-binding domain superfamily
Similarity search - Domain/homology
FE3-S4 CLUSTER / FLAVIN-ADENINE DINUCLEOTIDE / HEME C / UBIQUINONE-10 / Fructose dehydrogenase cytochrome subunit / Fructose dehydrogenase small subunit / Fructose dehydrogenase large subunit
Similarity search - Component
Biological speciesGluconobacter japonicus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsFukawa, E. / Miyata, T. / Makino, F. / Adachi, T. / Suzuki, Y. / Tanaka, H. / Namba, K. / Sowa, K. / Kitazumi, Y. / Shirai, O.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP22ama121003 Japan
Japan Society for the Promotion of Science (JSPS)JP21H01961 Japan
Japan Society for the Promotion of Science (JSPS)JP22K14831 Japan
CitationJournal: Electrochim Acta / Year: 2024
Title: Structural and electrochemical elucidation of biocatalytic mechanisms in direct electron transfer-type D-fructose dehydrogenase.
Authors: Fukawa, E. / Suzuki, Y. / Adachi, T. / Miyata, T. / Makino, F. / Tanaka, H. / Namba, K. / Sowa, K. / Kitazumi, Y. / Shirai, O.
History
DepositionDec 9, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 22, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Fructose dehydrogenase large subunit
B: Fructose dehydrogenase small subunit
C: Fructose dehydrogenase cytochrome subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,9159
Polymers132,1153
Non-polymers3,8006
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Fructose dehydrogenase ... , 3 types, 3 molecules ABC

#1: Protein Fructose dehydrogenase large subunit


Mass: 59755.285 Da / Num. of mol.: 1 / Mutation: N1190A
Source method: isolated from a genetically manipulated source
Details: fdhL / Source: (gene. exp.) Gluconobacter japonicus (bacteria) / Gene: fdhL / Production host: Gluconobacter oxydans (bacteria) / References: UniProt: M1VMF7
#2: Protein Fructose dehydrogenase small subunit / Fructose dehydrogenase subunit III


Mass: 20106.732 Da / Num. of mol.: 1 / Mutation: N1190A
Source method: isolated from a genetically manipulated source
Details: fdhS / Source: (gene. exp.) Gluconobacter japonicus (bacteria) / Gene: fdhS / Production host: Gluconobacter oxydans (bacteria) / References: UniProt: M1VB40
#3: Protein Fructose dehydrogenase cytochrome subunit / Fructose dehydrogenase subunit II


Mass: 52252.652 Da / Num. of mol.: 1 / Mutation: N1190A
Source method: isolated from a genetically manipulated source
Details: fdhC / Source: (gene. exp.) Gluconobacter japonicus (bacteria) / Gene: fdhC / Production host: Gluconobacter oxydans (bacteria) / References: UniProt: M1V1V5

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Non-polymers , 4 types, 6 molecules

#4: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE / Flavin adenine dinucleotide


Mass: 785.550 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#5: Chemical ChemComp-F3S / FE3-S4 CLUSTER / Iron–sulfur cluster


Mass: 295.795 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe3S4
#6: Chemical ChemComp-HEC / HEME C / Heme C


Mass: 618.503 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C34H34FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-U10 / UBIQUINONE-10 / Coenzyme Q10 / Coenzyme Q10


Mass: 863.343 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C59H90O4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: D-fructose dehydrogenase (N1190A) from Gluconobacter japonicus
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.14 MDa / Experimental value: YES
Source (natural)Organism: Gluconobacter japonicus (bacteria)
Source (recombinant)Organism: Gluconobacter oxydans (bacteria)
Buffer solutionpH: 6
Buffer component
IDConc.NameFormulaBuffer-ID
1176 mmol / LSodium dihydrogen phosphateNaH2PO41
224 mmol / LSodium hydrogen phosphateNa2HPO41
31.5 mmol / L2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanolC14H22O(C2H4O)n1
41 mmol / L2-MercaptethanolC2H6OS1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 60000 X / Calibrated magnification: 56754 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER / Temperature (max): 80 K / Temperature (min): 80 K / Residual tilt: 0.01 mradians
Image recordingAverage exposure time: 3 sec. / Electron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6843
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.0particle selection
2SerialEM3.9image acquisition
4cryoSPARC4.2.0CTF correction
7Cootmodel fitting
9cryoSPARC4.2.0initial Euler assignment
10cryoSPARC4.2.0final Euler assignment
11cryoSPARC4.2.0classification
12cryoSPARC4.2.03D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3435583
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 64345 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0098835
ELECTRON MICROSCOPYf_angle_d1.2812078
ELECTRON MICROSCOPYf_dihedral_angle_d9.2341241
ELECTRON MICROSCOPYf_chiral_restr0.0511276
ELECTRON MICROSCOPYf_plane_restr0.011568

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