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- EMDB-30809: Cryo-EM structure of DfgA-B at 2.54 angstrom resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-30809
TitleCryo-EM structure of DfgA-B at 2.54 angstrom resolution
Map data
Sample
  • Complex: DfgA-B
    • Protein or peptide: DfgA
    • Protein or peptide: DfgB
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 speciesEubacterium cellulosolvens (bacteria) / [Eubacterium] cellulosolvens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.54 Å
AuthorsMori T / Moriya T / Adachi N / Kawasaki M / Senda T / Abe I
Funding support Japan, 1 items
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 20, 2020-
Header (metadata) releaseDec 8, 2021-
Map releaseDec 8, 2021-
UpdateDec 8, 2021-
Current statusDec 8, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0464
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0464
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7dre
  • Surface level: 0.0464
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7dre
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30809.png

Downloads & links

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Map

FileDownload / File: emd_30809.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.676 Å
Density
Contour LevelBy AUTHOR: 0.0464 / Movie #1: 0.0464
Minimum - Maximum-0.10996941 - 0.21715085
Average (Standard dev.)-3.9332226e-05 (±0.006022702)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 324.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6760.6760.676
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z324.480324.480324.480
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.1100.217-0.000

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Supplemental data

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Mask #1

Fileemd_30809_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_30809_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_30809_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DfgA-B

EntireName: DfgA-B
Components
  • Complex: DfgA-B
    • Protein or peptide: DfgA
    • Protein or peptide: DfgB

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Supramolecule #1: DfgA-B

SupramoleculeName: DfgA-B / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: heterodimer complex of DfgA and DfgB
Source (natural)Organism: Eubacterium cellulosolvens (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 250 KDa

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Macromolecule #1: DfgA

MacromoleculeName: DfgA / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: [Eubacterium] cellulosolvens (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MYRYEKKGPK RGVALYSYSA EFGLTKTLED CFEDLHDMGA HGIEILANTH IENYPYPTDE WVEKWWRLCD KYEIVPVEYG NWIDSHVLGD RDLTTEESVE MLKRDIRLAH RLGFTVMRTK MPVINDLLEP VENWKEIIKG ALPLAEELGI KMCPEIHTPS NLKGKLVNDF ...String:
MYRYEKKGPK RGVALYSYSA EFGLTKTLED CFEDLHDMGA HGIEILANTH IENYPYPTDE WVEKWWRLCD KYEIVPVEYG NWIDSHVLGD RDLTTEESVE MLKRDIRLAH RLGFTVMRTK MPVINDLLEP VENWKEIIKG ALPLAEELGI KMCPEIHTPS NLKGKLVNDF VEFIKETGTK NFGLNIDFSV FRTSFAEGEW VDPNYTPNKP EDIIPLLPYV YCCHAKFIHM SDDFKETTIP YEEVVKTMED NGYEGYLLSE YEGADKYDEG YEVGQTLRKH HILLKNLLGD

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Macromolecule #2: DfgB

MacromoleculeName: DfgB / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: [Eubacterium] cellulosolvens (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MEKQVIQSVG FRNIKNGNGE ITGFQFKVKL PYYRGVFLSQ IRPGTLFVDG QKIEKDQITW TINGEEYTNQ EMRGDFKTHW ATTKPAVLKV KMPGGLAQGY HDLKYGFCFT SSYMPPIIQD GLDPDKESMV YMPEFGHHVN ERRLLIVKEE AAALEHHHHH H

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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

Concentration2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chlorideSodium Chloride
20.0 mMC8H18N2O4SHEPES
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: The grid was washed by acetone prior to use.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time was 20 seconds (blot force 0).
DetailsThis sample was mono-disperse.

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Electron microscopy

MicroscopeTFS TALOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 150000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1664 / Average exposure time: 62.06 sec. / Average electron dose: 50.0 e/Å2

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Image processing

Particle selectionNumber selected: 330866
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: OTHER
Details: An ab initio model was generated using RELION3's own implementation of Stochastic Gradient Descent (SGD) algorithm and low-pass filtered to 23.02 A for use as an initial model for 3D classification.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final 3D classificationNumber classes: 2 / Avg.num./class: 37186 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.54 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 60587
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

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

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