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- EMDB-2755: Cryo-electron microscopy of TibC dodecamer -

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Basic information

Entry
Database: EMDB / ID: EMD-2755
TitleCryo-electron microscopy of TibC dodecamer
Map dataReconstruction of TibC dodecamer
Sample
  • Sample: Complex of TibC
  • Protein or peptide: TibC
KeywordsBacterial autotransporters / Glycosyltransferase / Bacterial pathogenesis / Cryo-EM / Enzyme complex
Biological speciesEscherichia coli ETEC H10407 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.5 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: 5.7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 5.7
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2755.png

emd_2755.png

emd_2755_1.png

Downloads & links

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Map

FileDownload / File: emd_2755.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of TibC dodecamer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.2 Å/pix.
x 240 pix.
= 287.04 Å		1.2 Å/pix.
x 240 pix.
= 287.04 Å		1.2 Å/pix.
x 240 pix.
= 287.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.196 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.7 / Movie #1: 5.7
Minimum - Maximum-3.6064837 - 12.166548730000001
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-120-120-120
Dimensions240240240
Spacing240240240
CellA=B=C: 287.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1961.1961.196
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z287.040287.040287.040
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ442626
MAP C/R/S123
start NC/NR/NS-120-120-120
NC/NR/NS240240240
D min/max/mean-3.60612.167-0.000

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Supplemental data

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Sample components

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Entire : Complex of TibC

EntireName: Complex of TibC
Components
  • Sample: Complex of TibC
  • Protein or peptide: TibC

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Supramolecule #1000: Complex of TibC

SupramoleculeName: Complex of TibC / type: sample / ID: 1000 / Oligomeric state: TibC forms a dodecamer of six dimers / Number unique components: 1
Molecular weightTheoretical: 555 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
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: 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
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 155,000 times magnification
DateOct 11, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 3000 / Average electron dose: 18 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTF correction of each particle
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.5 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 8546

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