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- EMDB-2756: Cryo-electron microscopy of TibC12-TibA6 octadecamer in averaged ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2756
TitleCryo-electron microscopy of TibC12-TibA6 octadecamer in averaged conformation
Map dataReconstruction of TibC12-TibA6 complex in averaged conformation
Sample
  • Sample: Complex of TibC12-TibA6 octadecamer
  • Protein or peptide: TibC
  • Protein or peptide: TibA
Biological speciesEscherichia coli ETEC H10407 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.7 Å
AuthorsYao Q / Lu QH / Wan XB / Song F / Xu Y / Zamyatina A / Huang N / Zhu P / Shao F
CitationJournal: Elife / Year: 2014
Title: A structural mechanism for bacterial autotransporter glycosylation by a dodecameric heptosyltransferase family.
Authors: Qing Yao / Qiuhe Lu / Xiaobo Wan / Feng Song / Yue Xu / Mo Hu / Alla Zamyatina / Xiaoyun Liu / Niu Huang / Ping Zhu / Feng Shao /
Abstract: A large group of bacterial virulence autotransporters including AIDA-I from diffusely adhering E. coli (DAEC) and TibA from enterotoxigenic E. coli (ETEC) require hyperglycosylation for functioning. ...A large group of bacterial virulence autotransporters including AIDA-I from diffusely adhering E. coli (DAEC) and TibA from enterotoxigenic E. coli (ETEC) require hyperglycosylation for functioning. Here we demonstrate that TibC from ETEC harbors a heptosyltransferase activity on TibA and AIDA-I, defining a large family of bacterial autotransporter heptosyltransferases (BAHTs). The crystal structure of TibC reveals a characteristic ring-shape dodecamer. The protomer features an N-terminal β-barrel, a catalytic domain, a β-hairpin thumb, and a unique iron-finger motif. The iron-finger motif contributes to back-to-back dimerization; six dimers form the ring through β-hairpin thumb-mediated hand-in-hand contact. The structure of ADP-D-glycero-β-D-manno-heptose (ADP-D,D-heptose)-bound TibC reveals a sugar transfer mechanism and also the ligand stereoselectivity determinant. Electron-cryomicroscopy analyses uncover a TibC-TibA dodecamer/hexamer assembly with two enzyme molecules binding to one TibA substrate. The complex structure also highlights a high efficient hyperglycosylation of six autotransporter substrates simultaneously by the dodecamer enzyme complex.
History
DepositionAug 13, 2014-
Header (metadata) releaseSep 10, 2014-
Map releaseOct 22, 2014-
UpdateOct 22, 2014-
Current statusOct 22, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.87
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.87
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2756.png

emd_2756_1.png

Downloads & links

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Map

FileDownload / File: emd_2756.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of TibC12-TibA6 complex in averaged conformation
Voxel sizeX=Y=Z: 1.778 Å
Density
Contour LevelBy AUTHOR: 3.87 / Movie #1: 3.87
Minimum - Maximum-13.65540886 - 22.060565950000001
Average (Standard dev.)0.0 (±0.99999988)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 320.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.7781.7781.778
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z320.040320.040320.040
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ442626
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-13.65522.061-0.000

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Supplemental data

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Sample components

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Entire : Complex of TibC12-TibA6 octadecamer

EntireName: Complex of TibC12-TibA6 octadecamer
Components
  • Sample: Complex of TibC12-TibA6 octadecamer
  • Protein or peptide: TibC
  • Protein or peptide: TibA

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Supramolecule #1000: Complex of TibC12-TibA6 octadecamer

SupramoleculeName: Complex of TibC12-TibA6 octadecamer / type: sample / ID: 1000 / Oligomeric state: One TibA monomer binds to one TibC dimer / Number unique components: 2
Molecular weightTheoretical: 727 KDa

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Macromolecule #1: TibC

MacromoleculeName: TibC / type: protein_or_peptide / ID: 1
Details: Ferric ions were attached to specific cysteine residues. Lys230 was substituted by alanine to generate the catalytically inactive mutant.
Number of copies: 12 / Oligomeric state: Dodecamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli ETEC H10407 (bacteria)
Molecular weightTheoretical: 46 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Gold / Recombinant plasmid: pACYCDuet

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Macromolecule #2: TibA

MacromoleculeName: TibA / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli ETEC H10407 (bacteria)
Molecular weightTheoretical: 29 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Gold / Recombinant plasmid: pGEX-6p-2

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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

Concentration1 mg/mL
BufferpH: 7.6 / Details: 10mM Tris-HCl, 100mM NaCl, 2mM DTT
GridDetails: Quantifoil R2.1, 300 mesh
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
Method: 10 ug/ml bacitracin (Sigma) was added to the purified protein to obtain monodispersed particles and make the orientation distribution more anisotropic. Blot for 4 sec using blotting force 2 before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 155,000 times magnification
DetailsEnergy filter turned-off
DateMay 1, 2013
Image recordingCategory: CCD / Film or detector model: OTHER / Average electron dose: 18 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTF parameters calculated from whole micrograph using CTFFIND3
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 9.7 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 53303

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