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- PDB-7dre: Cryo-EM structure of DfgA-B at 2.54 angstrom resolution -

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Basic information

Entry
Database: PDB / ID: 7dre
TitleCryo-EM structure of DfgA-B at 2.54 angstrom resolution
Components
  • DfgB
  • Sugar phosphate isomerase/epimerase
KeywordsBIOSYNTHETIC PROTEIN / C-deglycosylase / sugar-isomerase-like
Function / homologyDomain of unknown function DUF6379 / Domain of unknown function (DUF6379) / Xylose isomerase-like, TIM barrel domain / Xylose isomerase-like TIM barrel / Xylose isomerase-like superfamily / isomerase activity / DUF6379 domain-containing protein / Sugar phosphate isomerase/epimerase
Function and homology information
Biological species[Eubacterium] cellulosolvens 6 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.54 Å
AuthorsMori, T. / Moriya, T. / Adachi, N. / Senda, T. / Abe, I.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP20am0101071 Japan
CitationJournal: Nat Commun / Year: 2021
Title: C-Glycoside metabolism in the gut and in nature: Identification, characterization, structural analyses and distribution of C-C bond-cleaving enzymes.
Authors: Takahiro Mori / Takuto Kumano / Haibing He / Satomi Watanabe / Miki Senda / Toshio Moriya / Naruhiko Adachi / Sanae Hori / Yuzu Terashita / Masato Kawasaki / Yoshiteru Hashimoto / Takayoshi ...Authors: Takahiro Mori / Takuto Kumano / Haibing He / Satomi Watanabe / Miki Senda / Toshio Moriya / Naruhiko Adachi / Sanae Hori / Yuzu Terashita / Masato Kawasaki / Yoshiteru Hashimoto / Takayoshi Awakawa / Toshiya Senda / Ikuro Abe / Michihiko Kobayashi /
Abstract: C-Glycosides, in which a sugar moiety is linked via a carbon-carbon (C-C) bond to a non-sugar moiety (aglycone), are found in our food and medicine. The C-C bond is cleaved by intestinal microbes and ...C-Glycosides, in which a sugar moiety is linked via a carbon-carbon (C-C) bond to a non-sugar moiety (aglycone), are found in our food and medicine. The C-C bond is cleaved by intestinal microbes and the resulting aglycones exert various bioactivities. Although the enzymes responsible for the reactions have been identified, their catalytic mechanisms and the generality of the reactions in nature remain to be explored. Here, we present the identification and structural basis for the activation of xenobiotic C-glycosides by heterocomplex C-deglycosylation enzymes from intestinal and soil bacteria. They are found to be metal-dependent enzymes exhibiting broad substrate specificity toward C-glycosides. X-ray crystallographic and cryo-electron microscopic analyses, as well as structure-based mutagenesis, reveal the structural details of these enzymes and the detailed catalytic mechanisms of their remarkable C-C bond cleavage reactions. Furthermore, bioinformatic and biochemical analyses suggest that the C-deglycosylation enzymes are widely distributed in the gut, soil, and marine bacteria.
History
DepositionDec 28, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 8, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Sugar phosphate isomerase/epimerase
B: DfgB
C: Sugar phosphate isomerase/epimerase
D: DfgB
E: Sugar phosphate isomerase/epimerase
F: DfgB
G: Sugar phosphate isomerase/epimerase
H: DfgB


Theoretical massNumber of molelcules
Total (without water)208,8908
Polymers208,8908
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16440 Å2
ΔGint-54 kcal/mol
Surface area58220 Å2

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Components

#1: Protein
Sugar phosphate isomerase/epimerase / DfgA


Mass: 33764.246 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) [Eubacterium] cellulosolvens 6 (bacteria)
Gene: EubceDRAFT1_2664 / Production host: Escherichia coli (E. coli) / References: UniProt: I5AX50
#2: Protein
DfgB


Mass: 18458.150 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) [Eubacterium] cellulosolvens 6 (bacteria)
Gene: EubceDRAFT1_2663 / Production host: Escherichia coli (E. coli) / References: UniProt: I5AX49

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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: DfgA and DfgB / Type: COMPLEX / Details: heterodimer complex of DfgA and DfgB / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.21 MDa / Experimental value: NO
Source (natural)Organism: [Eubacterium] cellulosolvens (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaClSodium chloride1
220 mMHEPESC8H18N2O4S1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono-disperse.
Specimen supportDetails: The grid was washed by acetone prior to use. / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K / Details: Blotting time was 20 seconds (blot force 0)

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 150000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 62.06 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1664

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1crYOLOparticle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7PHENIX1.18.2-3874model fittingMaptomodel
9RELION3initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.18.2-3874model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 330866
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60587 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00512920
ELECTRON MICROSCOPYf_angle_d0.55417488
ELECTRON MICROSCOPYf_dihedral_angle_d19.84724
ELECTRON MICROSCOPYf_chiral_restr0.0451900
ELECTRON MICROSCOPYf_plane_restr0.0042216

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